We investigated the effects of growth factors on the proliferation and matrix synthesis of anterior cruciate ligament fibroblasts. Fibroblasts from the anterior cruciate ligaments of dogs were transferred at the second passage in a defined medium. Epidermal growth factor, platelet-derived growth factor-AB, transforming growth factor-β1, insulin-like growth factor-1, and insulin, combined two by two following a 5 × 5 logarithmic concentration matrix, were added. Tridimensional curves showing cell proliferation at 24 hours against the concentration of two effectors were obtained for each combination. Collagen and proteoglycan productions were quantified using [14 C]glycine and Na2[35S]O4. Ratios of type I:III collagen and hydrodynamic size distributions of proteoglycans were assayed, respectively, by sodium dodecyl sulfate-polyacrylamidel gel electrophoresis and gel filtration chromatography. Epidermal growth factor had an effect nearly equivalent to that of platelet-derived growth factor-AB on cell proliferation. Both had a greater effect than insulin-like growth factor-1, which in turn had a greater effect than both the effect of insulin or the nearly equivalent effect of transforming growth factor-β1. Neither platelet-derived growth factor-AB nor insulin has a significant effect by itself on collagen production. Epidermal growth factor slightly decreases collagen production as well as the type I:III collagen ratio: both transforming growth factor-β1 and insulin-like growth factor-1 increase the same parameters. Epidermal growth factor inhibits the stimulation induced by transforming growth factor-β1. Similarly, insulin decreases the response to insulin-like growth factor-1. Proteoglycan production was significantly increased by all growth factors in this study, with transforming growth factor-β1 having the strongest effect, Small hydrodynamic size of proteoglycan was correlated to a high level of proteoglycan biosynthesis. The results may be readily applied to tissue engineering or provide a basis for in vivo investigations.