The antigenic cross-reactivity between Treponema pallidum and several pathogenic members of the family Spirochaetaceae was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting techniques. Blots of T. pallidum antigens were incubated with antiserum from rabbits infected or immunized with T. pallidum, Treponema paraluiscuniculi, Treponema hyodysenteriae (strains B204 and T22), Borrelia hermsii serotype 7, or Leptopsira interrogans serogroup Canicola. T. pallidum contained 22 antigenic molecules ranging from 85,000 to 12,000 daltons which were recognized by serum from rabbits infected with T. pallidum. Serum from rabbits infected with T. paraluiscuniculi cross-reacted with 21 of these molecules and faintly reacted with a band at 15,000 daltons which was not recognized by anti-T. pallidum serum. Antisera directed against strains B204 and T22 of T. hyodysenteriae cross-reacted with 11 and 10 antigens of T. pallidum, respectively. B. hermsii and L. interrogans serogroup Canicola antisera detected 11 and 10 treponemal antigens, respectively. Many of the T. pallidum antigens detected by antisera against T. hyodysenteriae, B. hermsii, or L. interrogans serogroup Canicola have been previously identified as containing moieties also found on the nonpathogenic Treponema phagedenis, biotype Reiter, and may therefore represent group antigens common to members of the family Spirochaetaceae.