The regulation of proteoglycan synthesis in a fibrocartilaginous tissue by mechanical loading was assessed in vitro. Discs of bovine tendon fibrocartilage were loaded daily with unconfined, cyclic, uniaxial compression (5 s/min, 20 min/day) and the synthesis of large and small proteoglycans was measured by incorporation of [35S]sulfate. All discs synthesized predominantly large proteoglycan when first placed in culture. After 2 weeks in culture nonloaded discs synthesized predominantly small proteoglycans whereas loaded discs continued to produce predominantly large proteoglycan. The turnover of 35S-labeled proteoglycan was not significantly altered by the compression regime. Increased synthesis of large proteoglycans was induced by a 4-day compression regime following 21 days of culture without compression. Inclusion of cytochalasin B during compression mimicked this induction. Autoradiography demonstrated that cell proliferation was minimal and confined to the disc edges whereas 35S-labeled proteoglycan synthesis occurred throughout the discs. These experiments demonstrate that mechanical compression can regulate synthesis of distinct proteoglycan types in fibrocartilage.