Because Rhizobium etli CE3 is normally dependent on an external source of biotin and lacks orthodox biotin biosynthesis genes, we undertook an analysis of biotin uptake in this organism. By complementation of a Sinorhizobium meliloti bioM mutant we isolated an R. etli chromosomal region encoding homologs of the S. meliloti bioMNB genes, whose products have been implicated in intracellular biotin retention in that organism. Disruption of the R. etli bioM resulted in a mutant which took up biotin at a lower rate and accumulated significantly less biotin than the wild type. As in S. meliloti, the R. etli bioMN gene-products resemble the ATPase and permease components, respectively, of an ABC-type transporter. The bioB gene product is in fact similar to members of the BioY family, which has been postulated to function in biotin transport, and we refer to this gene as bioY. An R. etli bioY mutant exhibited lower biotin uptake than the wild-type, providing the first experimental evidence for a role of BioY in biotin transport. We show that the bioMNY operon is transcriptionally repressed by biotin. An analysis of the competitiveness of the wild-type strain versus the bioM mutant showed that the mutant had a diminished capacity to form nodules on bean plants.