A clonal cell line exhibiting many of the properties of skeletal muscle has been derived from embryonic BDIX rat heart tissue. Multinucleated myotubes, formed by fusion of mononucleated myoblasts, simultaneously contract and produce regenerative action potentials in response to electrical stimulation or iontophoretic application of acetylcholine. The acetylcholine response is inhibited by 1−3 × 10⁻⁷ M d-tubocurarine, 10⁻⁷ g/ml α-neurotoxin or 1 × 10⁻⁴ M atropine. The specific activities of the enzymes myokinase and creatine phosphokinase (CPK) increase 3-fold and 20-fold, respectively, after myotube formation, but only CPK activity parallels the extent of fusion. Exponentially dividing myoblasts synthesize a predominantly brain-type CPK isoenzyme while fused myotubes synthesize a muscle type CPK isoenzyme. Electron microscopic analysis reveals that the myotubes contain elaborately branched tubular systems and numerous bundles of thick filaments with distinct M-bands. Some of the thick filament bundles are associated with thin filaments and organized into sarcomere-like structures with faint Z-regions, but no distinct Z-bands are observed.