Vinculin is a specialized protein with a molecular weight of 130 kilodaltons that is localized to focal regions of cell-to-substrate attachment. It is located precisely at the termini of actin-containing intracytoplasmic stress fibers, which appear to insert into the inner surface of the cell membrane via the vinculin junctions. By double-labeling corneal epithelial cells in tissue culture simultaneously for vinculin and actin, the intimate association of stress fibers and cell-to-substrate adhesion foci was demonstrated. At the leading edge of the migrating epithelial sheet, discrete vinculin patches were numerous in the direction of cell movement, while vinculin was less polarized and often diffusely distributed in the cytoplasm in the less motile cells toward the center of the cell colony. This study shows that corneal epithelial cell migration and spreading during wound healing is characterized by relatively firm substrate attachments at the advancing edge, with the cells behind the edge possibly being pulled forward via cytoskeletal contractile mechanisms.