The expression of alpha v beta3 integrin was examined in human skeletal muscle cells grown in vitro. The alpha v and beta3 subunits showed different patterns of expression during myogenesis. Expression of the alphav subunit did not change significantly during the growth and differentiation of muscle cells, whereas expression of the beta3 subunit was markedly down-regulated at both the message and protein levels. Down-regulation of beta3 subunit expression did not occur when cultures were treated with 5-bromo-2'-deoxyuridine to inhibit myoblast terminal differentiation, but did occur in cultures in which fusion was selectively inhibited by growth in EGTA-containing medium. These results suggest that the regulation of integrin beta3 subunit expression is tightly coupled to the myogenic terminal differentiation program, but is not simply a consequence of membrane reorganization due to the fusion process. Several stably transfected lines of mouse C2 myoblasts were derived from cultures transfected with a cDNA encoding the complete human beta3 integrin sequence under the control of the CMV promoter. Lines which constitutively expressed high levels of the human beta3 integrin subunit did not fuse or biochemically differentiate, whereas lines expressing moderate levels of the beta3 integrin subunit showed delayed fusion and differentiation. Lines expressing very low to undetectable levels of the human beta3 integrin subunit exhibited unimpaired fusion and differentiation. Taken together these results suggest (i) that down-regulation of alpha v beta3 integrin normally occurs as part of the myogenic terminal differentiation program, (ii) that this is mediated by regulation of the expression of the beta3 subunit, and (iii) that down-regulation of expression of the beta3 integrin subunit appears essential for myoblast differentiation.