We have previously demonstrated that cells of Treponema pallidum freshly extracted from infected rabbit testes can be intrinsically radiolabeled with [35 S]methionine to very high specific activities. In this study we used the inhibition of [35 S]methionine incorporation into trichloroacetic acid-precipitable protein in vitro as an assay to test the susceptibilities of three different pathogenic treponemal strains to various antibiotics. In general, the results correlated very well with the known efficacies of these antibiotics in treating human patients with syphilis. One of the strains tested, however, a clinical isolate of T. pallidum designated street strain 14, was found to exhibit high-level resistance to erythromycin and a closely related macrolide, roxithromycin (RU 965). Street strain 14 was originally isolated from a human patient with active secondary syphilis who failed to respond to erythromycin therapy. Thus, our results indicate that an erythromycin-resistant strain of T. pallidum can be responsible for erythromycin treatment failure. In addition, street strain 14 treponemes were found to be generally less susceptible by this assay to a variety of antibiotics than were treponemes of the T. pallidum Nichols strain. These findings suggest that the outer envelope of street strain 14 treponemes may be generally less permeable to antibiotics than is that of Nichols strain treponemes.