Cells from embryonic chicken muscle were cultivated in serum-free medium. After two days, the suspended cells (almost all of which were nondividing myoblasts) were subcultured in serum-containing medium, either in gelatincoated tissue culture dishes (to promote reattachment) or in bacteriological dishes (to prevent reattachment). The extent of fusion was high in both suspended and reattached cultures.Newly synthesized proteins from day-5 cultures were resolved by two-dimensional electrophoresis and detected by autoradiography. Not Most normal cell types exhibit anchorage-dependent proliferation; by contrast, many transformed cell lines proliferate well in suspension, a characteristic that appears to be consistently correlated with tumorigenicity (1, 2). For 3T6 cells, which show strict anchorage dependence of proliferation, forced suspension has been shown to result in a rapid decrease in both mRNA synthesis and mRNA degradation and in a slower decrease in total protein synthesis (3). Some proteins may be synthesized in near-normal amounts in suspension (4) and some mRNA species, and the proteins they code for, are synthesized in greater-than-normal amounts during recovery after reattachment (4). For these cells, then, there is reason to believe that their pattern of gene expression is regulated by cell-substrate interactions.Myogenic precursor cells from avian or mammalian embryos proliferate readily in monolayer culture. With time, they give rise to nondividing, terminally differentiating "myoblasts" that synthesize and accumulate muscle-specific molecules and form syncytial "myotubes." These cells elongate, elaborate the specialized subcellular structures characteristic of functional muscle, and contract (for review, see refs. 5-7).The influence of the culture substrate on muscle differentiation has received much attention (5). In clonal cultures myotube formation is favored by collagen-coated substrates. At high density this requirement is overcome, probably by cellderived collagen (5). Holtzer et al. (7) state that myogenic cells "remain alive when cultured on a surface to which they are unable to stick but will not differentiate unless they become attached." On the other hand, it has been reported that, under conditions that prevent normal cell-substrate interaction, myogenic cells did not divide and migrate, but were able to fuse and to synthesize and assemble myofibrillar proteins (8). Recently, several authors have observed that chicken myogenic cells can fuse in suspension, forming multinucleated "myoballs" (9-12). These myoballs possess acetylcholine receptors, generate action potentials, contract, and, when allowed to attach to a substrate, elongate into myotubes (10,11).In this paper, we reexamine the question of the anchorage dependence of skeletal muscle differentiation. Samples of the same cell suspension are cultivated in the same medium either in suspension or attached to culture dishes and then compared directly with respect to: (i) extent of fusion; (ii) pattern of synthesized prote...