Geobacter sulfurreducens, a representative of the family Geobacteraceae that predominates in Fe(III)-reducing subsurface environments, can grow by coupling the oxidation of hydrogen to the reduction of a variety of electron acceptors, including Fe(III), fumarate, and quinones. An examination of the G. sulfurreducens genome revealed two operons, hya and hyb, which appeared to encode periplasmically oriented respiratory uptake hydrogenases. In order to assess the roles of these two enzymes in hydrogen-dependent growth, Hya- and Hyb-deficient mutants were generated by gene replacement. Hyb was found to be required for hydrogen-dependent reduction of Fe(III), anthraquinone-2,6-disulfonate, and fumarate by resting cell suspensions and to be essential for growth with hydrogen and these three electron acceptors. Hya, in contrast, was not. These findings suggest that Hyb is an essential respiratory hydrogenase in G. sulfurreducens.