The role of fibronectin (FN) and vitronectin (VN) receptors for cell adhesion and matrix assembly was analyzed during human fetal myogenesis in vivo and in vitro. In human fetal muscle at 10 weeks gestational age FN and laminin are present in the extracellular matrix. Analysis of the integrin repertoire at this developmental stage reveals that the differentiated muscle cells in vivo express alpha 5 and alpha 6 integrins, but not alpha v, alpha 1, and alpha 3 integrins. However, in vitro cultured myoblasts (G6) isolated from the same gestational age express alpha v, alpha 1, and alpha 3 integrins in addition to alpha 5 and alpha 6 integrins. A more detailed analysis of FN and VN receptors in vitro shows that the localization of different alpha v heterodimers into focal contacts is differently regulated. Alpha v beta 1, and alpha v beta 3, are present at focal contacts throughout in vitro myogenesis whereas alpha v beta 5 appears to depend on an endogenously produced factor to localize to focal contacts. The alpha v beta 1, alpha v beta 5, and alpha 3 beta 1 heterodimers, often reported not to focalize, did form focal contacts in G6 cells, indicating that these myoblasts possess components facilitating the formation of cytoskeletal linkages containing these integrins. Alpha 5 beta 1 colocalized with FN in myoblast cultures, whereas myotubes lacked both FN and alpha 5 beta 1 on the cell surface. In summary, we show that concomitant with in vitro differentiation of G6 cells, FN matrix contacts are abolished, but vitronectin receptors continue to fulfill an anchoring function during the differentiation process in vitro. Further studies are needed to assess the relative importance of the FN and VN binding integrins for the differentiation process in comparison with the laminin-binding integrins alpha 6 and alpha 7, also present on these cells.