Abstract. Nitrous oxide (N₂O) is a trace gas that contributes to the greenhouse effect and stratospheric ozone depletion. The N₂O yield from nitrification (moles N₂O-N produced per mole ammonium-N consumed) has been used to estimate marine N₂O production rates from measured nitrification rates and global estimates of oceanic export production. However, the N₂O yield from nitrification is not constant. Previous culture-based measurements indicate that N₂O yield increases as oxygen (O₂) concentration decreases and as nitrite (NO₂⁻) concentration increases. Here, we have measured yields of N₂O from cultures of the marine β-proteobacterium Nitrosomonas marina C-113a as they grew on low-ammonium (50 μM) media. These yields, which were typically between 4 × 10⁻⁴ and 7 × 10⁻⁴ for cultures with cell densities between 2 × 10² and 2.1 × 10⁴ cells ml⁻¹, were lower than previous reports for ammonia-oxidizing bacteria. The observed impact of O₂ concentration on yield was also smaller than previously reported under all conditions except at high starting cell densities (1.5 × 10⁶ cells ml⁻¹), where 160-fold higher yields were observed at 0.5% O₂ (5.1 μM dissolved O₂) compared with 20% O₂ (203 μM dissolved O₂). At lower cell densities (2 × 10² and 2.1 × 10⁴ cells ml⁻¹), cultures grown under 0.5% O₂ had yields that were only 1.25- to 1.73-fold higher than cultures grown under 20% O₂. Thus, previously reported many-fold increases in N₂O yield with dropping O₂ could be reproduced only at cell densities that far exceeded those of ammonia oxidizers in the ocean. The presence of excess NO₂⁻ (up to 1 mM) in the growth medium also increased N₂O yields by an average of 70% to 87% depending on O₂ concentration. We made stable isotopic measurements on N₂O from these cultures to identify the biochemical mechanisms behind variations in N₂O yield. Based on measurements of δ¹⁵N^bulk, site preference (SP = δ¹⁵N^α−δ¹⁵N^β), and δ¹⁸O of N₂O (δ¹⁸O-N₂O), we estimate that nitrifier-denitrification produced between 11% and 26% of N₂O from cultures grown under 20% O₂ and 43% to 87% under 0.5% O₂. We also demonstrate that a positive correlation between SP and δ¹⁸O-N₂O is expected when nitrifying bacteria produce N₂O. A positive relationship between SP and δ¹⁸O-N₂O has been observed in environmental N₂O datasets, but until now, explanations for the observation invoked only denitrification. Such interpretations may overestimate the role of heterotrophic denitrification and underestimate the role of ammonia oxidation in environmental N₂O production.