To elucidate the importance of oligopeptide permease for Borrelia burgdorferi, the agent of Lyme disease, a chromosomal locus in B. burgdorferi that encodes homologues of all five subunits of oligopeptide permease has been identified and characterized. B. burgdorferi has multiple copies of the gene encoding the peptide-binding component, OppA; three reside at the chromosomal locus and two are on plasmids. Northern analyses indicate that each oppA gene is independently transcribed, although the three chromosomal oppA genes are also expressed as bi- and tri-cistronic messages. Induction of one of the plasmid-encoded oppA genes was observed following an increase in temperature, which appears to be an important cue for adaptive responses in vivo. The deduced amino acid sequences suggest that all five borrelial oppA homologues are lipoproteins, but the protease-resistance of at least one of them in intact bacteria is inconsistent with outer-surface localization. Insertional inactivation of a plasmid-encoded oppA gene demonstrates that it is not essential for growth in culture.