We report the first measurements of coupled nitrogen (N) and oxygen (O) isotopic variations of nitrate (NO3-) during its assimilation by laboratory cultures of marine phytoplankton and derive the N and O kinetic isotope effects for nitrate assimilation by three species of diatoms (Thalassiosira weissflogii, Thalassiosira oceanica, and Thalassiosira pseudonana) and a coccolithophorid (Emiliana huxleyi). Large interspecies and intraspecies variations in the N isotope effects were observed. The O isotope effect associated with nitrate consumption was consistently close to the N isotope effect, such that the 18O/16O and 15N/14N of nitrate varied in a ratio of ∼1:1, regardless of species or of the magnitude of the isotope effect. In addition, the 18O/16O and 15N/14N of internal nitrate of T. weissflogii grown under various environmental conditions were elevated relative to the medium nitrate by a proportion of ∼1: 1. These findings are consistent with a nitrate isotopic fractionation mechanism that involves nitrate reduction as the chief fractionating step. The observed N:O isotopic coupling during nitrate assimilation suggests that combined N and O isotopic measurements of water column nitrate can provide new constraints on the ocean N cycle.