Respiratory nitrate reductase (EC 1.7.99.4) from the denitrifying bacterium Pseudomonas stutzeri, strain ZoBell (ATCC 14405), was purified by a rapid three-step procedure of differential centrifugation, DEAE-Sepharose chromatography and gel filtration. The enzyme was released from the cytoplasmic membrane by heat. Purification was about 25-fold to an average specific activity of 27 µmol nitrate reduced per min per mg protein. Non-denaturing electrophoresis in the presence of 1% Triton X-100 resolved two catalytically active enzyme species with apparent Mr 140000 and 132000. The two forms had each a 112 kDa subunit and one or two types of small subunit (60 or 46 kDa). The membrane-bound enzyme was composed of two subunits of Mr 112000 and 60000 as shown by Western blot analysis. The subunit heterogeneity was caused by an endogenous proteinase which modified the small subunit. Nitrate reductase contained per Mr 172000 about 13 iron-sulfur groups and one gatom of molybdenum bound to a pterin cofactor. The electronic spectrum of the purified enzyme had a weak maximum at 410 nm; however, there was no spectral evidence for a cytochrome moiety. The Km value for nitrate was 3.8 mM. Azide and cyanide were inhibitory for nitrate reductase with Ki values of 0.7 µM and 0.1 mM, respectively. Addition of 0.1 mM azide to the growth medium increased the cell-free enzyme activity 4-fold, due to a concomitant overproduction of nitrate reductase.