Measurements of the stable isotope ratios of nitrogen (¹⁵N/¹⁴N) and oxygen (¹⁸O/¹⁶O) in nitrate (NO₃^–) enable identification of sources, dispersal, and fate of natural and contaminant NO₃^– in aquatic environments. The ¹⁸O/¹⁶O of NO₃^– produced by nitrification is often assumed to reflect the proportional contribution of oxygen atom sources, water, and molecular oxygen, in a 2:1 ratio. Culture and seawater incubations, however, indicate oxygen isotopic equilibration between nitrite (NO₂^–) and water, and kinetic isotope effects for oxygen atom incorporation, which modulate the NO₃^– 18O/¹⁶O produced during nitrification. To investigate the influence of kinetic and equilibrium effects on the isotopic composition of NO₃^– produced from the nitrification of ammonia (NH₃), we incubated streamwater supplemented with ammonium (NH₄⁺) and increments of ¹⁸O-enriched water. Resulting NO₃^– 18O/¹⁶O ratios showed (1) a disproportionate sensitivity to the ¹⁸O/¹⁶O ratio of water, mediated by isotopic equilibration between water and NO₂^–, as well as (2) kinetic isotope discrimination during O atom incorporation from molecular oxygen and water. Empirically, the NO₃^– 18O/¹⁶O ratios thus produced fortuitously converge near the ¹⁸O/¹⁶O ratio of water. More elevated NO₃^– 18O/¹⁶O values commonly reported in soils and oxic groundwater may thus derive from processes additional to nitrification, including NO₃^– reduction.