A reliable method for the primary culture of undifferentiated skeletal muscle cells is a prerequisite for the success of therapeutic strategies for Duchenne and Becker muscular dystrophies involving gene therapy. We have developed conditions for the long-term culture of both dystrophic and normal mouse muscle explants and have now successfully cultured both dystrophic and nondystrophic muscle satellite cells continuously for up to 18 months with minimal loss of stem cell phenotype and retention of the expression of muscle cell markers and the ability to fuse at high serum levels. Optimal culture conditions depend on both the age of the animal and the type of muscle explanted, but the majority of skeletal muscle explants produce large numbers of satellite cells within 4-10 days of explanting when cultured in Dulbecco's modified Eagle's medium/Ham's F12 medium supplemented with high levels of fetal calf serum (10-20%). A small proportion of explants will produce outgrowth when placed into serum-free medium and assay of the conditioned medium from these explants shows that they release large amounts of FGF-like activity(s) when compared to nonoutgrowing explants. This process can be augmented by the addition of acidic, but not basic, FGF. Cultures of both dystrophic and nondystrophic muscle grow predominantly as monomorphic rounded cells which stain positively with antibodies specific for skeletal fast muscle actin, myosin, and desmin. In the absence of substantial fibroblast cell contamination, these cells frequently form end-to-end connections and, under permissive conditions, they will fuse to form characteristic myotubes. A major difference observed between dystrophic and normal skeletal muscle explants was the reduction in fibroblast-like cell outgrowth of dystrophic explants.