We report the first measurements of coupled nitrogen (N) and oxygen (O) isotope fractionation of nitrate by laboratory cultures of denitrifying bacteria. Two seawater strains (Pseudomonas stutzeri, Ochrobactrum sp.) and three freshwater strains (Paracoccus denitrificans, Pseudomonas chlororaphis, Rhodobacter sphaeroides) were examined. Among four strains of facultative anaerobic denitrifiers, N and O isotope effects were variable, ranging from 5% to 25%, with evidence for a drop in the isotope effects as nitrate concentrations approached the half-saturation constant for nitrate transport. O isotope effects were similar to their corresponding N isotope effect, such that the progressive increase in nitrate δ18O, when plotted against that in δ15N (where δ18 Osample = [(18O : 16O)sample/(18O : 16O)reference - 1] x 1000, and δ15N sample = [(15N : 14N)sample/( 15N : 14N)reference - 1] x 1000), yielded slopes of 0.86 to 1.02, with a mean value of 0.96. R. sphaeroides, a photo-heterotroph that possesses only a periplasmic (nonrespiring) dissimilatory nitrate reductase, showed less variability in nitrate N isotope effects, between 13% and 20%, with a modal value of ∼15%. In contrast to the respiratory denitrifiers, R. sphaeroides consistently showed a distinct ratio of δ18O to δ15N change of ∼0.62. We hypothesize that heavy N and O isotope discrimination during respiratory denitrification occurs during the intracellular reduction of nitrate by the respiratory nitrate reductase, and the observed magnitude of fractionation is likely regulated by the ratio of cellular nitrate efflux relative to uptake. The data for R. sphaeroides are consistent with isotope discrimination directly reflecting the N and O isotope effects of the periplasmic nitrate reductase NAP, without modification by nitrate uptake and efflux. © 2008, by the American Society of Limnology and Oceanography, Inc.