The isotopic composition (δ15N) of nitrite produced during nitrate respiration by both growing and washed cells of the marine bacterium,Serratia marinorubra,was determined. In both the growing and washed cells, δ15N of the nitrite changed considerably with time. At least two reaction steps, producing different isotope effects (active transport of nitrate across membranes and reduction of nitrate to nitrite), appear to be involved. With washed cells, a fractionation factor as high as 1.039 was obtained—the highest ever reported for biologicalnitrate reduction. The physiological state of nitrate‐respiring bacteria in oxygen‐depleted subsurface waters of the sea is discussed from the viewpoint of isotope fractionation.