Remarkably, a number of definitive epithelia, such as that of the anterior lens, give rise when suspended within 3D gels of type I collagen, to elongate, bipolar shaped cells that exhibit the ultrastructure, polarity, and migratory ability of mesenchymal cells. They begin producing type I collagen and stop producing crystallins, type IV collagen, and laminin. Here, we investigated changes in beta 1 integrins and their extracellular matrix (ECM) ligands during this transdifferentiation. The former free surface of the lens epithelium that is now in contact with collagen begins within a day to stain intensely for beta 1 and it is this surface rather than the surface facing the basement membrane that gives rise to mesenchymal cells. Immunoprecipitation experiments reveal a large increase in the beta 1 integrin subunit on mesenchymal cells as compared to the epithelium of origin. The alpha 5 integrin subunit, which is barely detectable in the lens, increases in the mesenchymal cells and alpha 3 continues to be expressed at about the same level as in the epithelium. alpha 6, the epithelial integrin subunit, and laminin, its ECM ligand, are not detected immunohistochemically or biochemically in the mesenchyme. Rather, the mesenchymal cells secrete abundant fibronectin, the major ECM ligand for alpha 5 beta 1. RGD peptides do not inhibit the transformation but antibodies to beta 1 do perturb the emigration of mesenchymal cells from the lens apical surface. We conclude that the beta 1 integrins newly expressed on the apical epithelial surface interact with the surrounding 3D collagen gel to help bring about this unusual epithelial-mesenchymal transition.