In Lyme disease spirochetes, the ospC gene encodes a 22.7-kDa protein referred to as either the pC or the OspC protein. Using a variety of electrophoretic approaches followed by Southern blotting and probing with oligonucleotide probes, we mapped the ospC gene to a circular 26-kb plasmid. The ospC gene represents the first gene to be mapped to a circular plasmid in Lyme disease spirochetes. The occurrence of this gene in isolates belonging to each of the three Lyme disease-associated species, Borrelia burgdorferi, Borrelia garinii, and the VS461 group, was evaluated. The ospC gene was found to occur in all 21 isolates tested from each of the three species. Differential hybridization with a series of ospC probes in both Northern (RNA) and Southern blot analyses demonstrated that there is sequence variability in the ospC gene among isolates. While the gene was found to be present in all isolates, not all actively transcribed the gene. Transcriptional start site analyses suggest that the gene may be under the control of multiple promoters that are highly similar in nucleotide sequence.