In vitro binding assays and inhibition of cell adhesion with monoclonal antibodies have implicated the integrin alpha 3 beta 1 as a receptor for a variety of extracellular ligands. However, reports of alpha 3 beta 1-ligand interactions are inconsistent, and transfection studies have suggested that alpha 3 beta 1 is not sufficient for cell attachment to ligands other than kalinin/laminin 5. We used immunofluorescence to study subcellular localization of the alpha 3A cytoplasmic domain variant in different cultured cell types. Using standard fixation and permeabilization methods, antibodies specific for alpha 3A 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 immunofluorescence revealed alpha 3A 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 alpha 3A 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, alpha 5 and alpha V 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 alpha 3A beta 1 responds to a broad spectrum of extracellular ligands. Time course comparisons of the recruitment of alpha subunits from different fibronectin receptors suggested that localization of alpha 3A beta 1 to fibronectin-induced focal contacts was independent of the recruitment of alpha 5 and alpha 4 integrins. However, other studies have shown that alpha 3A beta 1 does not mediate initial cell adhesion to many of the ligands that induced its focal contact localization, including fibronectin. Therefore, we suggest that alpha 3A beta 1 may be a secondary receptor with post-cell-adhesion functions for a broad spectrum of extracellular matrices.