The membrane-bound hydrogenase from autotrophically cultured Rhizobium japonicum cells has been purified and partially characterized. During the purification process it was necessary to exclude O_2, add reducing agents and include the non-ionic detergent Genapol in buffers in order to maintain activity and prevent the enzyme frjom precipitation. The purified enzyme retained 78% of its activity after storage under H_2 at —80℃ for six months. The most highly purified fraction with a specific activity of 142 μmol methylene blue reduced·min~(-1)·mg protein~(-1), showed two major bands on SDS polyacrylamide electrophoresis gels with molecular weights of about 60,000 and 30,000. There was no positive correlation between hydrogenase specific activity and cytochrome b content of fractions obtained from the different steps in the purification process. No cytochrome b could be detected in the fraction with the highest specific activity. Some other characteristics of the hydrogenase from autotrophically cultu The membrane-bound hydrogenase from autotrophically cultured Rhizobium japonicum cells has been purified and partially characterized. During the purification process it was necessary to exclude O_2, add reducing agents and include the non-ionic detergent Genapol in buffers in order to maintain activity and prevent the The purified enzyme was retained 78% of its activity after storage under H 2 at -80 ° C for six months. The most highly purified fraction with a specific activity of 142 μmol methylene blue reduced · min -1 (mg · protein) ~ (-1), showed two major bands on SDS polyacrylamide electrophoresis gels with molecular weights of about 60,000 and 30,000. There was no positive correlation between hydrogenase specific activity and cytochrome b content of the different steps in the purification process. No cytochrome b could be detected in the fraction with the highest specific activity. Some other characteristics of the hydrogenase from a utotrophically cultu