We have utilized a model system to investigate myotrophic factors released by normal adult mouse muscles following a crush injury. We found that saline extracts from gently crushed mouse muscles (CME) contain potent mitogenic activities which act on primary newborn mouse myoblast cultures, as well as on mouse C2 cells, a mouse myoblast cell line. We compared the activity of CME on mouse myoblasts with that of basic fibroblast growth factor (bFGF) and insulin-like growth factor I (IGF-I), two growth factors known to be mitogenic for primary myoblasts (Allen, Dodson, and Lutein: Exp. Cell. Res., 152:154–160, 1984; DiMario and Strohman: Differentiation, 39:42–49, 1988; Allen and Boxhorn: J. Cell. Physiol., 138:311–315, 1989; Dodson, Allen, and Hossner: Endocrinology, 117:2357–2363, 1985; Florini and Magri: Am. J. Physiol., 256:C701–C711, 1989). We found that CME could act in an additive fashion to saturating doses of bFGF to increase proliferation in myoblast cultures. Additionally, CME acted additively to the combination of saturating amounts of bFGF and IGF-I on both C2 and primary myoblast cultures. We also examined additivity of CME with the combination of saturating doses of bFGF, IGF-I, transferrin (Tf), platelet-derived growth factor (PDGF), epidermal growth factor (EGF), adrenocorticotrophin (ACTH), and macrophage colony-stimulating factor (M-CSF). Our data indicate that CME contains Tf, as well as one or more uncharacterized mitogens for myoblasts which are distinct from Tf, the IGFs, bFGF, EGF, PDGF, M-CSF, and ACTH. These uncharacterized mitogens may act independently of known growth factors to stimulate myoblast proliferation, or may act through modulation of known growth factor activities. © 1992 Wiley-Liss, Inc.