We report a novel method for measurement of the oxygen isotopic composition (¹⁸O/¹⁶O) of nitrate (NO₃^-) from both seawater and freshwater. The denitrifier method, based on the isotope ratio analysis of nitrous oxide generated from sample nitrate by cultured denitrifying bacteria, has been described elsewhere for its use in nitrogen isotope ratio (¹⁵N/¹⁴N) analysis of nitrate.¹Here, we address the additional issues associated with ¹⁸O/¹⁶O analysis of nitrate by this approach, which include (1) the oxygen isotopic difference between the nitrate sample and the N₂O analyte due to isotopic fractionation associated with the loss of oxygen atoms from nitrate and (2) the exchange of oxygen atoms with water during the conversion of nitrate to N₂O. Experiments with ¹⁸O-labeled water indicate that water exchange contributes less than 10%, and frequently less than 3%, of the oxygen atoms in the N₂O product for Pseudomonas aureofaciens. In addition, both oxygen isotope fractionation and oxygen atom exchange are consistent within a given batch of analyses. The analysis of appropriate isotopic reference materials can thus be used to correct the measured ¹⁸O/¹⁶O ratios of samples for both effects. This is the first method tested for ¹⁸O/¹⁶O analysis of nitrate in seawater. Benefits of this method, relative to published freshwater methods, include higher sensitivity (tested down to 10 nmol and 1 μM NO₃^-), lack of interference by other solutes, and ease of sample preparation.