Abstract Autoradiographic analysis of Con A-stimulated cells cultured in syphilitic sera indicated that depression of [ 3H]thymidine incorporation reflected decreased numbers of cells incorporating radiolabeled thymidine, rather than decreased utilization of [ 3H]thymidine by all cells in the Con A responding population. Syphilitic sera which inhibited normal lymphocyte transformation induced by Con A or periodate oxidation did not inhibit [ 3H]thymidine incorporation by nonlymphoid cells. Syphilitic serum must be present during approximately the first 12 hr of lymphocyte stimulation in order to inhibit incorporation of [ 3H]thymidine. The inhibitory factor(s) remained stable for 24 hr at 37 °C, and was not significantly removed by absorption with Treponema pallidum, rabbit red blood cells, or rabbit peripheral blood lymphocytes. Peripheral blood lymphocytes obtained from different normal rabbit donors exhibited variable sensitivity to the same serum sample. Theoretically, the inhibitory factor(s) found in syphilitic serum may act to reduce lymphocyte reactivity induced during acute orchitis, or to inhibit lymphocyte responsiveness to T. pallidum itself.