We analyzed expression of a putative oligopeptide permease (Opp) of Borrelia burgdorferi. Unlike the opp operons of other bacteria for which there is a single substrate binding protein, B. burgdorferi codes for three substrate binding proteins (OppA-I to -III) in its opp operon and an additional two homologs on plasmids (OppA-IV and -V). Instead of a single promoter region regulating transcription of the entire operon, as seen in other bacterial opp operons, it appears that among oppA-I, -II, and -III, as well as oppA-IV and -V, each has a potential upstream promoter region. We tested the function of these putative promoter sequences by fusion to a promoterless beta-galactosidase reporter gene in pCB182. Each of the promoter regions was found to be active. The level of activity in the reporter constructs closely paralleled the level of expression of each gene in in vitro-grown B. burgdorferi. Changes in carbon and nitrogen availability differentially affected individual promoters, but no changes in promoter activity were seen when Escherichia coli bacteria (with the promoter constructs) were grown in various concentrations of phosphate and leucine and changes in pH. Expression of specific oppA genes with B. burgdorferi varied significantly between its mouse and fed and unfed tick hosts. Differences in regulation of opp gene expression suggest a potential role in environmental response by the organism.