The major antigens of Treponema pallidum, Nichols strain were examined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting techniques. Organisms were extracted from rabbit testes, purified by differential centrifugation, washed, and disrupted by sonication before solubilization in SDS buffer, SDS-PAGE analysis on 12.5% gels revealed at least 35 distinguishable polypeptides with molecular weights between 14,000 and 100,000. Proteins were electrophoretically transferred from gels to nitrocellulose paper and were stained with pooled rabbit anti-T. pallidum serum (IRS) and 125I-protein A. At least eight distinct antigenic molecules were revealed, with the major component residing at 48,000 daltons. Extensive washing of T. pallidum before solubilization did not alter the antigenic profile. Five molecules with common treponemal antigenic determinants were identified both by staining blots of T. phagedenis, biotype Reiter with IRS and by staining blots of T. pallidum with rabbit anti-Reiter antiserum. Three putative T. pallidum-specific antigens were identified by staining T. pallidum with IRS that had been extensively adsorbed with Reiter treponemes.