Rhizobium japonicum hydrogenase was purified to homogeneity from soybean root nodules by four column chromatography steps after solubilization from membranes by treatment with a nonionic detergent. The specific activity was from 40 to 65 mumol H2 oxidized min-1 mg protein-1 and was increased 450-fold relative to that in bacteroids. The yield of activity was from 7 to 12%. The molecular weight of the native enzyme was 104,000 as determined by sucrose density gradient centrifugation. Electrophoresis in the presence of sodium dodecyl sulfate revealed two subunits with molecular weights of 64,000 and 35,000, indicating an alpha beta subunit structure. The amino acid content of the protein indicated 20 cysteine residues. Analysis of the metal content indicated 0.59 +/- 0.06 mol Ni/mol hydrogenase and 6.5 +/- 1.2 mol Fe/mol hydrogenase. Antisera prepared to the hydrogenase cross-reacted with the enzyme in bacteroid extracts at all stages of the purification but did not cross-react with extracts of Alcaligenes eutrophus grown under chemolithotrophic conditions. The similarity of rhizobial hydrogenase to the particulate hydrogenases of A. eutrophus and A. latus is discussed.