Rhizobium meliloti transports succinate, fumarate, malate, and aspartate by means of the dicarboxylate transport system, which is encoded by dct genes located on the exo megaplasmid. Analysis of these genes using Tn5 insertion mutagenesis revealed three complementation groups within a 5.9-kb HindIII fragment. The sequence of this fragment and the sites of Tn5 insertion were determined. Three genes, dctA, dctB, and dctD, were identified as the only three open-reading frames in locations consistent with the complementation data. The dctA gene is preceded by the sequence CTGGCACG-N4-TTGCT, which is characteristic of promoters requiring the ntrA-encoded protein for activation. The dctA-encoded protein is highly hydrophobic and contains eight potential transmembrane helices, indicating that it is probably the structural component of the transport system responsible for movement of dicarboxylates from the periplasm across the inner membrane. The dctB and dctD genes are transcribed in the opposite direction to dctA. They encode proteins with homology to the R. leguminosarum bv. viceae dicarboxylate transport proteins regulating expression of dctA and to other proteins comprising two-component regulatory systems. The dctB-encoded protein includes a putative periplasmic N-terminal domain that senses the presence of dicarboxylates and a C-terminal cytoplasmic domain that activates the dctD-encoded protein. The C-terminus of the dctD-encoded protein shows homology to several DNA-binding proteins, indicating that it is probably the domain which binds DNA in the dctA promoter region to regulate dctA transcription. All the R. meliloti mutants altered in dctA, dctB, and dctD were complemented by the dct region from R. l. bv. viceae.