Actin, a major protein involved in muscle cell contraction, is also associated with cell motility, cell-substrate adhesion, and cell-shape changes in non-muscle cells. By electrophoresis and scanning densitometry, actin was found to constitute about 4% to 6% of the total cellular protein in the human corneal epithelium. The fluorescent probe, NBD-phallacidin, which specifically binds to filamentous actin (F-actin), was used to demonstrate the distribution of this protein in cultured corneal epithelial cells obtained from human eyebank eyes. Actin was present in the cytoplasm in two, often coexisting, patterns: (1) within numerous parallel and convergent linear bundles known as stress fibers, and (2) diffusely in the cytoplasm, with a cortical region of increased density in the peripheral cytoplasm adjacent to the plasmalemma. Actin has been implicated in the generation of cellular movement forces during the migration phase of corneal epithelial healing. Stress fibers, however, may not be absolutely necessary for the generation of the actual locomotive forces, but may function instead in anchoring the cell to the substrate and in elaborating cell-shape changes during cell spreading.