The cytochrome b subunit (HydC) of Wolinella succinogenes hydrogenase binds two haem B groups. This is concluded from the haem B content of the isolated hydrogenase and is confirmed by the response of its cytochrome b to redox titration. In addition, three of the four haem B ligands were identified by characterizing mutants with the corresponding histidine residues replaced by alanine or methionine. Substitution in HydC of His-25, His-67 or His-186, which are, in addition to His-200, predicted to be haem B ligands, caused the loss of quinone reactivity of the hydrogenase, while the activity of benzylviologen reduction was retained. The corresponding mutants did not grow with H2 as electron donor and either fumarate or polysulphide as terminal electron acceptor. The mutants grown with formate and fumarate did not catalyse electron transport from H2 to fumarate or to polysulphide, or quinone reduction by H2, in contrast to the wild-type strain. Cytochrome b was not reduced by H2 in the Triton X-100 extract of the mutant membranes, which contained wild-type amounts of the mutated HydC protein. Substitution in HydC of His-122, His-158 or His-187, which are predicted not to be haem B ligands, yielded mutants with wild-type properties. Substitution in HydA of His-188 or of His-305 resulted in mutants with the same properties as those lacking one of the haem B ligands of HydC. His-305 is located in the membrane-integrated C-terminal helix of HydA. His-188 of HydA is predicted to be a ligand of the distal iron-sulphur centre that may serve as the direct electron donor to the haem B groups of HydC. The results suggest that each of the three predicted haem B ligands of HydC tested (out of four) is required for electron transport from H2 to either fumarate or polysulphide, and for quinone reactivity. This also holds true for the two conserved histidine residues of HydA.