Abstract The intact hydrogenase from Desulfovibrio vulgaris (Miyazaki) was purified and characterized. The purified hydrogenase had a molecular weight of 92.0 kDa, with two different subunits (Mw = 62.5 kDa and 29.5 kDa). The molecular weight of the large subunit of the intact hydrogenase was 2 kDa larger than the tryptic digested hydrogenase. The absorption spectrum and the EPR spectrum of the intact hydrogenase were identical to those of tryptic digested hydrogenase. Phase separation study indicated the detergent solubilized hydrogenase was more hydrophobic than tryptic digested hydrogenase.