The weight ratio of myosin/actin, the myosin heavy chain content as the percentage of total protein (wt/wt), and the kinds of myosin light chains were determined in (a) standard muscle cultures, (b) pure myotube cultures, and (c) fibroblast cultures. Cells for these cultures were obtained from the breast of 11-day chick embryos. Standard cultures contain, in addition to myotubes, large numbers of replicating mononucleated cells. By killing these replicating cells with cytosine arabinoside, pure myotube cultures were obtained. The myosin/actin ratio (wt/wt) for pure myotube, standard muscle, and fibroblast cultures average 3.1, 1.9, and 1.1 respectively. By day 7, myosin in myotube cultures represents a minimum of 7% of the total protein, but about 3% in standard cultures and less than 1.5% in fibroblasts cultures. Myosin from standard cultures contains light chain LC1, LC2, and LC3, with a relative stoichiometry of the molarity of 1.0:1.9:0.5 and mol wt of 25,000, 18,000 and 16,000 daltons, identical to those in adult fast muscle. Myosin from pure myotubes exhibits light chains LC1 and LC2, with a molar ratio of 1.5:1.6. Myosin from fibroblast cultures possesses two light chains with a stoichiometry of 1.8:1.8 and mol wt of 20,000 and 16,000 daltons. Clearly, the faster migrating light chain, LC3, found in standard cultures is synthesized not by the myotubes but ty the mononucleated cells. In myotubes, both the assembly of the sarcomeres and the interaction between thick and thin filaments required for spontaneous contraction occur in the absence of light chain LC3. One set of structural genes for the myosin light and heavy chains appears to be active in mononucleated cells, whereas another set appears to be active in multinucleated myotubes.