The hydrogenase solubilized from the particulate fraction from Desulfovibrio vulgaris Miyazaki F (IAM 12604) has been crystallized. Although the solubilized hydrogenase purified by the previous method (Yagi, T., Kimura, K., Daidoji, H., Sakai, F., Tamura, S., and Inokuchi, H. (1976) J. Biochem. (Tokyo) 79,661-671) revealed a single band upon disc electrophoresis, it could not be crystallized. The apparently homogeneous hydrogenase has been separated into three components of similar molecular weights by high performance liquid chromatography on DEAE-Toyopearl. Each hydrogenase component was successfully crystallized by means of the vapor diffusion method with polyethylene glycol or 2-methyl-2,4-pentanediol as a precipitating agent. Seeding procedure is necessary to grow an x-ray grade crystal. Preliminary x-ray experiments reveal that crystals grown from one component are in space group of P2(1)2(1)2(1) with a = 102.1(1), b = 126.8 (3), and c = 66.9(1) A. The unit cell volume of 8.66 X 10(5) A3 suggests that it contains one molecule/asymmetric unit (Vm = 2.43). The crystals grown from another component are in the same space group with a = 99.6(1), b = 126.8(3), c = 66.9(1) A, and the unit cell volume is 8.45 X 10(5) A3 (Vm = 2.37). The crystals diffract more than 2.5 A and are suitable for complete crystal analysis. Up to 4 A resolution native data have been collected on a diffractometer.