The Bacillus subtilis genome contains two tandem genes, ebrA and ebrB, which encode two homologues of the SMR family of multidrug efflux transporters. The sequences of EbrA and EbrB are highly similar to each other and to that of EmrE, the prototypical SMR transporter of Escherichia coli. Drug resistance profiling and drug binding experiments showed that the presence of both EbrA and EbrB is required for proper transport function. EbrA and EbrB directly interact and combine to form a functional transporter. They likely form a heterodimer in analogy to the EmrE homodimer. Mutagenesis experiments indicate that the conserved membrane-embedded glutamates in the first transmembrane helices of both EbrA and EbrB are required for multidrug efflux activity. However, the two glutamates are nonequivalent since EbrA E15 is required for substrate binding while EbrB E14 is not. Our studies support a model in which functional residues in EbrAB are relegated to at least two sets that participate in distinct steps of the active drug transport process.