Wolinella succinogenes can grow by anaerobic respiration with fumarate or polysulfide as the terminal electron acceptor, and H-2 or formate as the electron donor. A Delta hydABC mutant lacking the hydrogenase structural genes did not grow with H-2 and either fumarate or polysulfide. In contrast to the wild-type strain, the mutant grown with fumarate and with formate instead of H-2 did not catalyze the reduction of fumarate, polysulfide, dimethylnaphthoquinone, or benzyl viologen by H-2. Growth and enzymic activities were restored upon integration of a plasmid carrying hydABC into the genome of the Delta hydABC mutant. The Delta hydABC mutant was complemented with hydABC operons modified by artificial stop codons in hydA (StopA) or at the 5'-end of hydC (StopC). The StopC mutant lacked HydC, and the hydrophobic C-terminus of HydA was missing in the hydrogenase of the StopA mutant. The two mutants catalyzed benzyl viologen reduction by H-2. The enzyme activity was located in the membrane of the mutants. A mutant with both modifications (StopAC) contained the activity in the periplasm. The three mutants did not grow with H-2 and either fumarate or polysulfide, and did not catalyze dimethylnaphthoquinone reduction by H2 We conclude that the same hydrogenase serves in the anaerobic respiration with fumarate and with polysulfide. HydC and the C-terminus of HydA appear to be required for both routes of electron transport and for dimethylnaphthoquinone reduction by H2. The hydrogenase is anchored in the membrane by HydC and by the C-terminus of HydA. The catalytic subunit HydB is oriented towards the periplasmic side of the membrane.