A serum-free medium has been devised that supports the proliferation and differentiation of primary cultures of rat skeletal muscle satellite cells for up to 4 d. The medium consists of a mixture of Dulbecco's modified Eagle's medium and MCDB-104 plus insulin, dexamethasone, pituitary fibroblast growth factor, Deutsch fetuin, and linoleic acid. In addition to promoting the formation of myotubes from satellite cells, a decrease in fibroblast contamination of these cultures was observed when cultures grown in serum-free medium were compared to cultures grown in serum-containing medium.