Washed cells of Desulfovibrio vulgaris strain Marburg oxidized H2, formate, lactate or pyruvate with sulfate, sulfite, trithionate, thiosulfate or oxygen as electron acceptor. CuCl2 as an inhibitor of periplasmic hydrogenase inhibited H2 and formate oxidation with sulfur compounds, and lactate oxidation in H2-grown, but not in lactate-grown cells. H2 oxidation was sensitive to O2 concentrations above 2% saturation. Carbon monoxide inhibited the oxidation of all substrates tested. Additions of micromolar H2 pulses to cells incubated in KCl in the presence of various sulfur compounds (reductant pulse method) resulted in a reversible acidification. This proton release was stimulated by thiocyanate, methyl triphenylphosphonium (MTPP+) or valinomycin plus EDTA, and completely inhibited by the uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP), CuCl2 or carbon monoxide. The extrapolated H+/H2 ratios obtained with sulfate, sulfite, trithionate or thiosulfate varied from 1.0 to 1.7. Micromolar additions of O2 to cells incubated in the presence of excess of electron donor (oxidant pulse method) caused proton translocation with extrapolated H+/H2 ratios of 3.9 with H2, 1.6 with lactate and 2.4 with pyruvate. Since a periplasmic hydrogenase can release at maximum 2 H+/H2, it is concluded that D. vulgaris is able to generate a proton gradient by vectorial proton translocation across the cytoplasmic membrane and by extracellular proton release by a periplasmic hydrogenase.