Nitrite is a central intermediate in the nitrogen cycle and can persist in significant concentrations in ocean waters, sediment pore waters, and terrestrial groundwaters. To fully interpret the effect of microbial processes on nitrate (NO₃^-), nitrite (NO₂^-), and nitrous oxide (N₂O) cycling in these systems, the nitrite pool must be accessible to isotopic analysis. Furthermore, because nitrite interferes with most methods of nitrate isotopic analysis, accurate isotopic analysis of nitrite is essential for correct measurement of nitrate isotopes in a sample that contains nitrite. In this study, nitrite salts with varying oxygen isotopic compositions were prepared and calibrated and then used to test the denitrifier method for nitrite oxygen isotopic analysis. The oxygen isotopic fractionation during nitrite reduction to N₂O by Pseudomonas aureofaciens was lower than for nitrate conversion to N₂O, while oxygen isotopic exchange between nitrite and water during the reaction was similar. These results enable the extension of the denitrifier method to oxygen isotopic analysis of nitrite (in the absence of nitrate) and correction of nitrate isotopes for the presence of nitrite in “mixed” samples. We tested storage conditions for seawater and freshwater samples that contain nitrite and provide recommendations for accurate oxygen isotopic analysis of nitrite by any method. Finally, we report preliminary results on the equilibrium isotope effect between nitrite and water, which can play an important role in determining the oxygen isotopic value of nitrite where equilibration with water is significant.