A new procedure for determining the susceptibility of Treponema pallidum subsp. pallidum to antimicrobial agents was developed, utilizing a tissue culture system which promotes the in vitro multiplication of this organism. In the absence of antibiotics, T. pallidum (Nichols virulent strain) multiplied an average of 10-fold when incubated for 7 days in the presence of Sf1Ep cottontail rabbit epithelial cell cultures. Varied concentrations of penicillin G, tetracycline, erythromycin, and spectinomycin were added to triplicate cultures to determine their effects on treponemal multiplication, motility, and virulence. The MIC of each antibiotic was defined as the lowest concentration which prevented treponemal multiplication, whereas the MBC was defined as the lowest concentration which abrogated the ability of the cultured treponemes to multiply and cause lesions in rabbits. The in vitro culture technique provided highly reproducible MICs and (in parentheses) MBCs of each of the antibiotics tested: aqueous penicillin G, 0.0005 (0.0025) microgram/ml; tetracycline, 0.2 (0.5) microgram/ml; erythromycin, 0.005 (0.005) microgram/ml; and spectinomycin, 0.5 (0.5) microgram/ml. The significance of these results in light of the in vivo activities and the previous in vitro evaluations of these antibiotics is discussed. The T. pallidum in vitro cultivation system shows promise as a method for studying the interaction between T. pallidum and antimicrobial agents and for screening new antibiotics for syphilis therapy.