Migrating corneal epithelial cells in culture elaborate lush arrays of actin filament bundles (stress fibers) which terminate peripherally at discrete cell-to-substratum adhesion complexes. These complexes contain several specific adhesion plaque proteins, of which vinculin is one. Vinculin, a 130 kD protein, putatively links the intracellular actin bundles in a transmembrane fashion (via integrin, a specific plasmalemmal junction protein) to focal attachment sites onto the underlying extracellular matrix substratum. Migrating rat corneal epithelial cells in tissue culture are treated with 0.1-1.0 microgram/ml cytochalasin D, an inhibitor of actin polymerization. This resulted in a generalized disruption of stress fibers, causing actin to the diffusely redistributed into discrete, round patches. Most, but not all, of these patches of actin appeared to colocalize with foci of vinculin. The focal patches of vinculin appeared to be larger and more diffuse in the cytochalasin-treated cells compared to the drug-free cells. These cytochalasin-induced cytoskeletal changes were associated with total cessation of cell migration and increased cellular detachment from the substratum.