Nitrite is an important intermediate species in the biogeochemical cycling of nitrogen, but its role in natural aquatic systems is poorly understood. Isotopic data can be used to study the sources and transformations of NO₂^- in the environment, but methods for independent isotopic analyses of NO₂^- in the presence of other N species are still new and evolving. This study demonstrates that isotopic analyses of N and O in NO₂^- can be done by treating whole freshwater or saltwater samples with the denitrifying bacterium Stenotrophomonas nitritireducens, which selectively reduces NO₂^- to N₂O for isotope ratio mass spectrometry. When calibrated with solutions containing NO₂^- with known isotopic compositions determined independently, reproducible δ¹⁵N and δ¹⁸O values were obtained at both natural-abundance levels (±0.2−0.5‰ for δ¹⁵N and ±0.4−1.0‰ for δ¹⁸O) and moderately enriched ¹⁵N tracer levels (±20−50‰ for δ¹⁵N near 5000‰) for 5−20 nmol of NO₂^- (1−20 μmol/L in 1−5 mL aliquots). This method is highly selective for NO₂^-and was used for mixed samples containing both NO₂^- and NO₃^- with little or no measurable cross-contamination. In addition, mixed samples that were analyzed with S. nitritireducens were treated subsequently with Pseudomonas aureofaciens to reduce the NO₃^- in the absence of NO₂^-, providing isotopic analyses of NO₂^- and NO₃^- separately in the same aliquot. Sequential bacterial reduction methods like this one should be useful for a variety of isotopic studies aimed at understanding nitrogen cycling in aquatic environments. A test of these methods in an agricultural watershed in Indiana provides isotopic evidence for both nitrification and denitrification as sources of NO₂^- in a small stream.