Because previous studies of three-dimensional skeletal muscle cultures have shown limited differentiation, the goal of this study was to establish conditions that would produce mature sarcomeres in a mammalian-derived skeletal muscle construct. We evaluated the differentiation of bioartificial muscles generated from C(2)C(12) myoblasts in a collagen gel cultured under steady, passive tension for up to 36 days. Staining for alpha-actinin, myosin, and F-actin indicated the presence of striated fibers as early as 6 days post-differentiation. Electron microscopy at 16 days post-differentiation revealed multinucleated myotubes with ordered, striated myofibers. At 33 days, the cultures contained collagen fibers and showed localization of paxillin at the fiber termini, suggesting that myotendinous junctions were forming. The present study demonstrates mature muscle synthesis in a three-dimensional system using a pure mammalian myoblast cell line. Our results suggest that this culture model can be used to evaluate the effects of various mechanical and biochemical cues on muscle development under normal and pathological conditions.