The relationship between Treponema pallidum infection and the synthesis of proteoglycans by organ cultures of rabbit testes was investigated. Two proteoglycan fractions, large (CL-6B Kav 0.05) and small (Kav 0.25), that were not synthesized at detectable levels by cultures from rabbits infected with T. pallidum for 0 or 5 days, were produced by cultures from 10-, 15- and 20-day infected rabbits. The small proteoglycan appeared to be synthesized first because greater amounts of this fraction were detected in extracts of cultures from 10-day infected animals. The large proteoglycan fraction may have been induced directly by T. pallidum because increased synthesis correlated with maximal treponemal numbers, 15 days after infection. In contrast, the induction of the smaller proteoglycan did not appear to be directly related to numbers of elutable organisms. The proteoglycans synthesized between 10 and 20 days after infection were analyzed for glycosaminoglycan (GAG) size and composition. The size of GAGs beta-eliminated from the proteoglycans generally increased over the 10-20-day infection period. Whereas the composition of the small proteoglycan fraction was largely unchanged during the 10-20-day period (45% chondroitin sulphate (CS), 55% dermatan sulphate (DS)), the amount of DS in the high molecular weight proteoglycan fraction decreased from 50-20% during this period (CS 50-80%). Autoradiography studies revealed that increased proteoglycan synthesis in T. pallidum-infected testes was localized to cells lining the seminiferous tubules and to fibroblasts infiltrating peritubular spaces.