<p>Nitrous oxide (N<span class="inline-formula">₂</span>O) is a potent greenhouse gas and ozone depletion agent, with a significant natural source from marine oxygen-deficient zones (ODZs). Open questions remain, however, about the microbial processes responsible for this N<span class="inline-formula">₂</span>O production, especially hybrid N<span class="inline-formula">₂</span>O production when ammonia-oxidizing archaea are present. Using <span class="inline-formula">¹⁵</span>N-labeled tracer incubations, we measured the rates of N<span class="inline-formula">₂</span>O production from ammonium (NH<span class="inline-formula"><math xmlns="http://www.w3.org/1998/Math/MathML" id="M6" display="inline" overflow="scroll" dspmath="mathml"><mrow><msubsup><mi/><mn mathvariant="normal">4</mn><mo>+</mo></msubsup></mrow></math><span><svg:svg xmlns:svg="http://www.w3.org/2000/svg" width="8pt" height="15pt" class="svg-formula" dspmath="mathimg" md5hash="3fe22ea21bb8c3940d1d54b092ea883d"><svg:image xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="bg-21-3215-2024-ie00001.svg" width="8pt" height="15pt" src="bg-21-3215-2024-ie00001.png"/></svg:svg></span></span>), nitrite (NO<span class="inline-formula"><math xmlns="http://www.w3.org/1998/Math/MathML" id="M7" display="inline" overflow="scroll" dspmath="mathml"><mrow><msubsup><mi/><mn mathvariant="normal">2</mn><mo>-</mo></msubsup></mrow></math><span><svg:svg xmlns:svg="http://www.w3.org/2000/svg" width="9pt" height="16pt" class="svg-formula" dspmath="mathimg" md5hash="f7a532d5ce2f1361c746623f9ea69b14"><svg:image xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="bg-21-3215-2024-ie00002.svg" width="9pt" height="16pt" src="bg-21-3215-2024-ie00002.png"/></svg:svg></span></span>), and nitrate (NO<span class="inline-formula"><math xmlns="http://www.w3.org/1998/Math/MathML" id="M8" display="inline" overflow="scroll" dspmath="mathml"><mrow><msubsup><mi/><mn mathvariant="normal">3</mn><mo>-</mo></msubsup></mrow></math><span><svg:svg xmlns:svg="http://www.w3.org/2000/svg" width="9pt" height="16pt" class="svg-formula" dspmath="mathimg" md5hash="7de8959157e6c258409d4c11688ca166"><svg:image xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="bg-21-3215-2024-ie00003.svg" width="9pt" height="16pt" src="bg-21-3215-2024-ie00003.png"/></svg:svg></span></span>) in the eastern tropical North Pacific ODZ and the isotopic labeling of the central (<span class="inline-formula"><i>α</i></span>) and terminal (<span class="inline-formula"><i>β</i></span>) nitrogen (N) atoms of the N<span class="inline-formula">₂</span>O molecule. We observed production of both doubly and singly labeled N<span class="inline-formula">₂</span>O from each tracer, with the highest rates of labeled N<span class="inline-formula">₂</span>O production at the same depths as the near-surface N<span class="inline-formula">₂</span>O concentration maximum. At most stations and depths, the production of <span class="inline-formula">⁴⁵</span>N<span class="inline-formula">₂</span>O<span class="inline-formula">^<i>α</i></span> and <span class="inline-formula">⁴⁵</span>N<span class="inline-formula">₂</span>O<span class="inline-formula">^<i>β</i></span> were statistically indistinguishable, but at a few depths there were significant differences in the labeling of the two nitrogen atoms in the N<span class="inline-formula">₂</span>O molecule. Implementing the rates of labeled N<span class="inline-formula">₂</span>O production in a time-dependent numerical model, we found that N<span class="inline-formula">₂</span>O production from NO<span class="inline-formula"><math xmlns="http://www.w3.org/1998/Math/MathML" id="M24" display="inline" overflow="scroll" dspmath="mathml"><mrow><msubsup><mi/><mn mathvariant="normal">3</mn><mo>-</mo></msubsup></mrow></math><span><svg:svg xmlns:svg="http://www.w3.org/2000/svg" width="9pt" height="16pt" class="svg-formula" dspmath="mathimg" md5hash="d615913ec88b34ee0c05b0f0374db64d"><svg:image xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="bg-21-3215-2024-ie00004.svg" width="9pt" height="16pt" src="bg-21-3215-2024-ie00004.png"/></svg:svg></span></span> dominated at most stations and depths, with rates as high as 1600 <span class="inline-formula">±</span> 200 pM N<span class="inline-formula">₂</span>O d<span class="inline-formula">⁻¹</span>. Hybrid N<span class="inline-formula">₂</span>O production, one of the mechanisms by which ammonia-oxidizing archaea produce N<span class="inline-formula">₂</span>O, had rates as high as 230 <span class="inline-formula">±</span> 80 pM N<span class="inline-formula">₂</span>O d<span class="inline-formula">⁻¹</span> that peaked in both the near-surface and deep N<span class="inline-formula">₂</span>O concentration maxima. Based on the equal production of <span class="inline-formula">⁴⁵</span>N<span class="inline-formula">₂</span>O<span class="inline-formula">^<i>α</i></span> and <span class="inline-formula">⁴⁵</span>N<span class="inline-formula">₂</span>O<span class="inline-formula">^<i>β</i></span> in the majority of our experiments, we infer that hybrid N<span class="inline-formula">₂</span>O production likely has a consistent site preference, despite drawing from two distinct substrate pools. We also found that the rates and yields of hybrid N<span class="inline-formula">₂</span>O production were enhanced at low dissolved oxygen concentrations ([O<span class="inline-formula">₂</span>]), with hybrid N<span class="inline-formula">₂</span>O yields as high as 20 % at depths where [O<span class="inline-formula">₂</span>] was below detection (880 nM) but nitrification was still active. Finally, we identified a few incubations with [O<span class="inline-formula">₂</span>] up to 20 <span class="inline-formula">µ</span>M where N<span class="inline-formula">₂</span>O production from NO<span class="inline-formula"><math xmlns="http://www.w3.org/1998/Math/MathML" id="M48" display="inline" overflow="scroll" dspmath="mathml"><mrow><msubsup><mi/><mn mathvariant="normal">3</mn><mo>-</mo></msubsup></mrow></math><span><svg:svg xmlns:svg="http://www.w3.org/2000/svg" width="9pt" height="16pt" class="svg-formula" dspmath="mathimg" md5hash="256fee4a33a420f3414d74c50d919438"><svg:image xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="bg-21-3215-2024-ie00005.svg" width="9pt" height="16pt" src="bg-21-3215-2024-ie00005.png"/></svg:svg></span></span> was still active. A relatively high O<span class="inline-formula">₂</span> tolerance for N<span class="inline-formula">₂</span>O production via denitrification has implications for the feedbacks between marine deoxygenation and greenhouse gas cycling.</p>