The somatomedin-like growth factors cartilage-derived factor (CDF) and multiplication-stimulating activity (MSA) stimulate DNA synthesis and proliferation of rabbit costal chondrocytes under serum-free conditions. Previously, we suggested that CDF and MSA act on chondrocytes in an early G1 phase to stimulate DNA synthesis. CDF and MSA have synergistic effects with epidermal growth factor (EGF) or fibroblast growth factor (FGF) in stimulating DNA synthesis of the cells. The mode of combined action of CDF or MSA with EGF or FGF in chondrocytes was studied by sequential treatments with these agents. EGF or FGF had synergistic effects with CDF or MSA in stimulating DNA synthesis, even when added 10 h after the latter. Synergism was also observed in cells pretreated with CDF or MSA; That is, the cultures were treated for 5 h with CDF or MSA and then washed, and treated with FGF or EGF. However, when CDF or MSA was added more than 5 h after EGF or FGF, no synergism of effects was observed. These findings suggest that the cultured chondrocytes become activated to interact with FGF or EGF for commitment to DNA synthesis when they are exposed to somatomedin-like growth factors at an early stage in the G1 phase. Thus chondrocytes are under a different mechanism of growth control from fibroblastic cells.