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Chick embryo myoblasts were fused in suspension culture to form myoballs by modification of previous procedures that excluded the use of divalent ion chelators and antimitotic drugs and included the continuous presence of serum in order to analyze the organized appearance and synthesis of major cytoskeletal proteins during cell attachment and spreading. The organization of the major cytoskeletal proteins actin, tubulin, and vimentin was assessed by fluorescence microscopy under these conditions as well as under conditions in which the myoballs were allowed to attach and spread on a collagen-coated substrate. Actin, detected by fluorescence microscopy, stained myoballs diffusely and was reorganized to form stress fibers in the attached and spreading myoball. Nuclei were segregated to a centrally located lattice of microtubules. The microtubule-specific drugs nocodazole and taxol prevented myoball spreading and the establishment of myotube polarity, respectively. Vimentin appeared as wavy ribbons in a perinuclear position around attached and spreading myoballs. In parallel studies, the synthesis of these cytoskeletal proteins was analyzed by radioisotopic labeling and polyacrylamide gel electrophoresis. These studies showed that myoballs possess altered ratios of actin and tubulin isoforms and of phosphorylated and nonphosphorylated vimentin compared to myotubes. These ratios rapidly change to the myotube pattern when myoballs are allowed to attach to solid substrata. Thus, although both myoballs and myotubes undergo muscle-specific differentiation, their cytoskeletal proteins are morphologically and biochemically distinct.