Nitrous oxide (N₂O) isotopocules provide key insights into microbial nitrogen cycling, but their interpretation requires well-constrained values for both oxygen isotope signatures (δ¹⁸O–N₂O) and intramolecular ¹⁵N site preference (SP) associated with N₂O production pathways. Site preference is widely used to distinguish N₂O formation pathways because bacterial denitrification is generally assumed to yield SP values near 0 ‰ through canonical NorB-mediated NO reduction. However, the extent to which SP remains stable across physiological states and changing NO reduction pathways remains poorly constrained. Likewise, interpretation of δ¹⁸O–N₂O associated with denitrification requires understanding the relative contributions of branching kinetic isotope effects and oxygen atom exchange between nitrite and water during N₂O formation.</p> <p>Here, we investigated N₂O isotopic signatures during denitrification by <em>Pseudomonas aureofaciens</em> (NirK-bearing) and <em>Pseudomonas chlororaphis</em> (NirS-bearing) under active-growth and resuspension conditions using quantum cascade laser absorption spectroscopy (QCLAS) and isotope ratio mass spectrometry (IRMS). SP tracked canonical NorB-mediated NO reduction but transiently increased above +10 ‰ during early N₂O production, indicating temporary activity of alternative NO reductases. These dynamics were only resolved through continuous QCLAS measurements, highlighting the importance of time-resolved isotopic observations. While SP remains a useful indicator of NO reduction mechanisms, these results show that even within denitrification, shifts between NO reduction pathways may lead to variable SP signatures.</p> <p>In parallel, we quantified oxygen atom exchange between nitrite and water using incubations prepared in natural-abundance and ¹⁸O-enriched water. Contrary to expectations from denitrifier-method studies, P. aureofaciens exhibited substantial and highly variable oxygen-atom exchange (38–100 %), far exceeding previously reported values (<9 %). In contrast, P. chlororaphis showed consistently high but less variable exchange (~66 %). Resuspension experiments reproduced the characteristic low- and high-exchange behavior reported for these strains under denitrifier-method conditions, demonstrating that these exchange values are specific to the methodological framework and not representative of actively growing systems. These results show that oxygen atom exchange is not governed solely by nitrite reductase identity (NirS vs. NirK) but is strongly modulated by physiological state and metabolic context. As a result, δ¹⁸O–N₂O cannot be interpreted as a fixed tracer of denitrification pathways outside the constrained conditions of the denitrifier method.</p> <p>Together, these findings suggest that denitrifying bacteria may generate N₂O with a broader range of δ¹⁸O–N₂O and SP than previously assumed. This calls for a reassessment of N₂O isotopocule interpretations and emphasizes the need to integrate isotopic measurements with physiological and biochemical constraints.