Clonally derived cultures of porcine skeletal muscle satellite cells were developed. The mitogenic effects of basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), insulin-like growth factors (IGF-I and -II), and platelet-derived growth factors (PDGF-AA and -BB) were examined. Individually, bFGF, IGFs, and PDGF-BB stimulated proliferation of porcine satellite cells grown in basal serum-free medium or Minimum Essential Medium containing 2% fetal bovine serum (MEM-2% FBS). EGF stimulated proliferation in MEM-2% FBS, but neither EGF nor PDGF-AA were mitogenic when added to serum-free medium. The interactions among bFGF, EGF, IGF-I, and PDGF-BB were examined in serum-free medium, using growth factor concentrations shown in dose-response experiments to induce maximal proliferative responses (10 ng/ml bFGF, EGF and PDGF-BB, and 50 ng/ml IGF-I). The combination of bFGF and IGF-I dramatically increased proliferation, and IGF-I also synergized with EGF to increase proliferation. EGF, IGF-I, and bFGF interacted with PDGF-BB to stimulate proliferation. With the exception of EGF and bFGF, combinations of two growth factors typically resulted in greater than additive responses. Simultaneous exposure of satellite cells to bFGF, PDGF-BB, EGF, and IGF-I produced a fivefold increase in DNA compared to cells grown in basal serum-free medium. Elimination of EGF did not reduce the mitogenic response, yet removal of IGF-I, bFGF, or PDGF-BB reduced proliferation by approximately 40, 20, and 10%, respectively. These mitogens are likely physiological regulators of porcine satellite cell activity.