In vitro binding assays and inhibition of cell adhesion with monoclonal antibodies have implicated the integrin α3β1 as a receptor for a variety of extracellular ligands. However, reports of α3β1-ligand interactions are inconsistent, and transfection studies have suggested that α3β1 is not sufficient for cell attachment to ligands other than kalinin/laminin 5. We used immunofluorescence to study subcellular localization of the α3A cytoplasmic domain variant in different cultured cell types. Using standard fixation and permeabilization methods, antibodies specific for α3A stained most cell types in a diffuse pattern, consistent with previous reports. Surprisingly, however, chemical cross-linking of integrins to the extracellular matrix and extraction of the cytoskeleton prior to immuno-fluorescence revealed α3A in focal contacts of most cells tested, suggesting that the cytoplasmic domain was concealed in intact focal contacts by cytoskeletal or other cytoplasmic proteins. The α3A subunit localized to focal contacts in several cell types cultured on fibronectin, kalinin/laminin 5, EHS-laminin/laminin 1, type IV collagen, or vitronectin. In contrast, α5 and αV integrins were detected in focal contacts only in cells grown on their known ligands (fibronectin, and fibronectin or vitronectin, respectively). Therefore, our results show that α3Aβ1 responds to a broad spectrum of extracellular ligands. Time course comparisons of the recruitment of α subunits from different fibronectin receptors suggested that localization of α3Aβ1 to fibronectin-induced focal contacts was independent of the recruitment of α5 and α4 integrins. However, other studies have shown that α3Aβ1 does not mediate initial cell adhesion to many of the ligands that induced its focal contact localization, including fibronectin. Therefore, we suggest that α3Aβ1 may be a secondary receptor with post-cell-adhesion functions for a broad spectrum of extracellular matrices.