Our goal was to determine the early response of corneal epithelial cells to living modified stromal substrates. We examined the distribution of integrin subunits (alpha 6 and beta 4), vinculin and the organization of F-actin in epithelial cells after cell-matrix and cell-cell hypothesized that the distribution of proteins in the cell matrix attachment complex would be altered if the substrate was modified. Integrin subunits, alpha 6 and beta 4, were chosen as they play a role in cell matrix adhesion and adhesion site formation. Corneal epithelial cells were cultured from explants and seeded on three corneal substrates (a stroma containing an intact basal lamina, a cornea lacking a basal lamina and a cornea treated with alkali). After 3 h of incubation, the tissue was fixed and stained with monoclonal antibodies specific for vinculin and for the integrin subunits alpha 6 and beta 4. The organization of F-actin was assessed using rhodamine phalloidin. The localization of the proteins was recorded with confocal laser scanning microscopy. Filamentous actin delineated the lateral cell membranes of corneal epithelial cells. The organization of actin and distribution of vinculin and integrin subunits of epithelial cells cultured on stromal substrates containing a basal lamina mimicked a simple epithelial organization. In contrast, when cells are cultured on the substrate lacking a basal lamina and alkaline treated corneal substrates the distribution of the specific proteins examined was altered. Vinculin and alpha 6 were present along membranes of cells cultured on substrates lacking a basal lamina and were diffuse in cells cultured on the alkaline substrates. These studies demonstrate that changes in the distribution of adhesion and cytoskeletal proteins in response to different surfaces may contribute to the healing dynamics in different wounds.