Thirty isolates of Borrelia burgdorferi sequentially cultured from 15 naturally infected white-footed mice (Peromyscus leucopus) were examined for variability in protein and plasmid profiles. Heterogeneity was detected in OspB and OspC and in proteins between 18.0 and 28.0 kDa by PAGE. Plasmid profiles were heterogeneous in the first isolate from 11 mice (73%) and between the first and last sequential isolate from 13 mice (87%). Comparison of the first and last isolates showed increased expression of OspC in 6 mice (40%) and was associated in each case with a shift in mobility of a 16.0-kb plasmid, suggesting that regulatory elements of ospC may reside on this plasmid. Hybridization studies suggested that individual mice may have been infected by a heterogeneous population of spirochetes and that changes in the protein and plasmid profiles between the first and last sequential isolates from some mice may have been the result of clonal selection.