Hydrogenase from Chlamydomonas reinhardtii was purified to homogeneity by five columnchromatography deps under strict anaerobic conditions. The cells were disrupted by mild treatment with detergent. The enzyme was purified 6100-fold, resulting in a specific activity for H, evolution of 935 pmol . min-' . mg protein-' at 25"C, using reduced methyl viologen as electron donor. The optimal temperature for hydrogen evolution is 60°C, the optimal pH value is 6.9. The K, value for methyl viologen is 0.83 mM, for ferredoxin, 35 pM. From SDSFAGE gels, the protein was judged to be pure. On non-denaturing gels, run under nitrogen, a single band was detected after activity staining. This band corresponded to the single band observed on denaturing SDS gels, which had an apparent molecular mass of 48 m a . If the band was cut out of the native gel and incubated with reduced methyl viologen, hydrogen evolution could be measured. The purified enzyme contains 4Fe atorns/mol. The amino acid composition and the N-terminal amino acid sequence (24 residues) of the protein were determined. No significant amino acid sequence homologies could be found to any sequences from prokaryotic hydrogenases.Many prokaryotes and several eukaryotes have in common an enzyme complex catalyzing the reversible reaction 2 H + + 2 e = H 2 .