Organ cultures of syphilitic and normal rabbit testes were incubated with 35S-sulfate for labeling of proteoglycans. Syphilitic rabbit testes synthesized three macromolecular fractions (I, II, and III) which were not detected in extracts of normal uninfected tissue. The three fractions comprised a larger (approximately 10(6) mol wt) chondroitin sulfate/dermatan sulfate proteoglycan (Fraction I), a smaller (approximately 10(5) mol wt) chondroitin sulfate/dermatan sulfate proteoglycan (Fraction II), and a putative sulfated glycoprotein of Mr 40 kd (Fraction III). The glycosaminoglycan chains of both proteoglycans eluted with a Kav of 0.45 on Sepharose CL-6B, consistent with a molecular weight of 25,000. The smaller proteoglycan was not a cleavage product of the larger species. Erythromycin had no significant effect on the synthesis of any of the three macromolecules. In contrast, the synthesis of both proteoglycans was totally inhibited by a 2-hour preincubation with cycloheximide, which suggests that the constitutive "pools" of the two core proteins were small. The putative sulfated 40-kd glycoprotein was insensitive to a 2-hour preincubation with cycloheximide.