The Gram-negative Deltaproteobacterium D. vulgaris Hildenborough is able to grow with sulfate, sulfite and thiosulfate as electron acceptors and in their absence via fermentation or syntrophic association with hydrogenotrophic organisms. Despite decades of research, the mechanism of energy generation by D. vulgaris is not well understood. Genome sequence revealed genes for at list six different hydrogenases, four periplasmic and two cytoplasmic. Although some of them have been characterized, their roles in D. vulgaris remain obscure. We have examined the consequences of mutations in two cytoplasmic hydrogenases on respiratory and syntrophic growth: 1) echA (DVU0434), the first gene in the operon for the Ech type NiFe- containing hydrogenase and 2) cool (DVU2288), the third gene in the operon coding for the second cytoplasmic NiFe-containing hydrogenase Coo. Growth rate, cell yield, and metabolite production were characterized for three growth conditions: i) sulfate with lactate or pyruvate, ii) sulfate with acetate and hydrogen, and iii) in syntrophic association with a hydrogenotrophic methanogen. Hydrogen oxidation activities in soluble and membrane fractions of the mutants and a wild type were not significantly different. Although growth rates of both mutants on sulfate with pyruvate or lactate were comparable to the wild type, hydrogen evolution was much greater for the echA mutant. Growth of the echA mutant was severely impaired relative to the wild type or cooL mutant with sulfate and hydrogen/CO2, but this mutation had little affect on syntrophic growth on either lactate or pyruvate. Syntrophic growth of the cooL mutant was severely impaired on lactate but not on pyruvate. Based on these observations we concluded that the main role of the Ech hydrogenase is in hydrogen oxidation. The Coo hydrogenase likely directly coupled the oxidation of lactate to pyruvate by accepting electrons and reducing protons inside the cells.