Two respiratory membrane-bound nitrate reductase (NR) isoenzymes, NRI and NRII, have been purified for the first time from one single microorganism. Triton X-100-solubilized NRs were purified by a three-step procedure of differential centrifugation, Q-Sepharose chromatography, and gel filtration on Sephacryl S-300. Both isoenzymes were purified to homogeneity by the criteria of NR activity staining in polyacrylamide gels run under non-denaturating conditions and coincident staining of the protein band by silver nitrate. NRI is composed of three subunits of 116 kDa, 68 kDa, and 56 kDa, whereas NRII is composed of four subunits of 116 kDa, 68 kDa, 59 kDa, and 56 kDa. The 116-kDa subunit of NRI and the 59-kDa subunit of NRII exhibited immunological cross-reactivity with the respiratory NR of Pseudomonas stutzeri strain ZoBell.