Syphilis is a chronic infection with early relapses that are hypothesized to result from the emergence of phenotypic variants ofTreponema pallidum. Recent studies demonstrated that TprK, a target of protective immunity, is heterogeneous in several T. pallidum strains, but not in Nichols strain Seattle (A. Centurion-Lara, C. Godornes, C. Castro, W. C. Van Voorhis, and S. A. Lukehart, Infect. Immun. 68:824–831, 2000). Analysis of PCR-amplified tprK from Nichols strain UNC and Street strain 14 treponemes showed that TprK has seven regions of intrastrain heterogeneity resulting from amino acid substitutions, insertions, and deletions. In contrast, analysis of PCR-amplified tprJshowed little intrastrain or interstrain heterogeneity. Reverse transcriptase PCR analysis demonstrated that mRNA transcripts representing unique polymorphic TprK proteins are present during syphilitic infection. Southern hybridization confirmed that Nichols strain UNC and Street strain 14 each contain a single copy oftprK, indicating that intrastrain heterogeneity is due to the presence of multiple treponemal subpopulations which contain a variant form of tprK.