Suspensions of embryonic chick leg muscle cells have been employed to establish replicate monolayer cultures. Such cultures grow rapidly to form a confluent layer of cells. Despite culture conditions generally assumed to be "dedifferentiative," a high degree of differentiation is attained in terms of the development of cross-striated myofibrils and contractility. To evaluate the possible role of in situ nuclear replication in the development of multinuclearity in muscle cells, an inhibitor of deoxyribonucleic acid (DNA) synthesis, methyl-bis (betachloroethylamine) (nitrogen mustard), was employed. Treatment with nitrogen mustard at concentration levels that profoundly inhibit DNA synthesis does not block the formation of multinuclear cells. On the basis of the pattern of nuclear enlargement after nitrogen mustard treatment, the cytologic picture of treated cultures is interpreted as indicating that the nuclei of only mononucleated cells are normally capable of proliferation. An absence of proliferative activity in the nuclei of multinuclear cell suggests that myoblast proliferation is self-limiting in this system and may explain, in part, the high degree of differentiation attained in monolayer culture.