ABSTRACTTwo unique isolates ofBorrelia burgdorferi, differing in plasmid content and outer surface protein C expression, were cultured on sequential captures of a single free-livingPeromyscus leucopusmouse and were examined for differences in transmissibility. Both isolates were transmissible from inoculated C.B-17 mice to larvalIxodes scapularisticks and, subsequently, from infected nymphal ticks to C3H/HeJ mice. Plasmid and protein analyses suggested that the original isolates were a mixed population ofB. burgdorferi, and cloning by limiting dilution resulted in the identification of two clonal groups. In addition to being heterogeneous in plasmid and genomic macrorestriction analyses, the clones varied with respect to the electrophoretic mobilities and antigenicity of their OspC proteins, as shown by their reactivity to a panel of monoclonal antibodies. Plasmid analysis of sequential isolates from C3H mice experimentally infected with the primary isolate or various mixtures of its subclones showed an apparently random fluctuation in clonal dominance in the majority of mice. Surprisingly, mice infected with each subclone were permissive to superinfection with the heterologous subclone, despite the presence of anti-B. burgdorferiantibodies at the time of the secondary challenge. These results show conclusively that mice captured at Lyme disease enzootic sites may be infected by mixed populations of genetically and antigenically distinctB. burgdorfericlones and that these infections can be acquired by coinfection or by sequential infection. The lack of cross-immunization between clones existing within a naturally occurring population may play a role in the maintenance of the genetic heterogeneity ofB. burgdorferiin nature.