The 26 to 28 kb circular plasmid of B. burgdorferi sensu lato (cp26) is ubiquitous among bacteria of this group and contains loci implicated in the mouse–tick transmission cycle. Restriction mapping and Southern hybridization indicated that the structure of cp26 is conserved among isolates from different origins and culture passage histories. The cp26 ospC gene encodes an outer surface protein whose synthesis within infected ticks increases when the ticks feed, and whose synthesis in culture increases after a temperature upshift. Previous studies of ospC coding sequences showed them to have stretches of sequence apparently derived from the ospC genes of distantly related isolates by homologous recombination after DNA transfer. We found conservation of the promoter regions of the ospC and guaA genes, which are divergently transcribed. We also demonstrated that the increase in OspC protein after a temperature upshift parallels increases in mRNA levels, as expected if regulatory regions adjoin the conserved sequences in the promoter regions. Finally, we used directed insertion to inactivate the ospC gene of a non‐infectious isolate. This first example of directed gene inactivation in B. burgdorferi shows that the OspC protein is not required for stable maintenance of cp26 or growth in culture.