In the absence of electron acceptors, many Desulfovibrio species grow on non-fermentable substrates via syntrophic association with hydrogen consuming methanogens. We examined the physiology of D. vulgaris Hildenborough growing syntrophically with Methanococcus maripaludis LL using a combination of transcriptional and deletion mutant analyses. Syntrophic cocultures were established in chemostats on minimal media amended with lactate but lacking electron acceptor. Replicated whole genome transcriptional analyses identified 169 and 254 genes that were significantly up- or down-regulated, respectively, relative to sulfate-limited monocultures grown at the same generation time. The majority of up-regulated genes were associated with energy production/conservation, signal transduction mechanisms, and amino acid transport/metabolism. A number of the down-regulated genes were associated with signal transduction mechanisms, inorganic ion transport/metabolism and amino acid transport and metabolism. In order to elucidate possible roles of several highly up-regulated genes associated with electron transfer and energy conservation, we constructed mutants of Desulfovibrio deleted in a subset of these genes. Cocultures developed with these mutants displayed a range of growth yields, implicating a putative carbon-monoxide induced hydrogenase (Coo, DVU2286-93) and a high-molecular weight cytochrome (Hmc, DVU0531-6) in energy conservation during syntrophic growth. Mutant monocultures grew to the same density on lactate/sulfate as the wildtype. The cooL and hmc mutants grew significantly slower and to approximately 25% yields of wildtype cocultures. Together, these data suggest a role of these genes in energy conservation of D. vulgaris Hildenborough during syntrophic growth.