Chronic use of proparacaine, a topical ocular anesthetic, is associated with punctate keratopathy and delayed epithelial wound healing. Spreading corneal epithelial cells normally elaborate cytoplasmic arrays of actin-rich stress fibers which insert onto the inner surface of the cell membrane at discrete adhesion complexes. As actin is implicated in cell-to-substratum adhesion and cell motility, the effects of proparacaine on the actin cytoskeleton of corneal epithelial cells were studied in vitro. Spreading rat corneal epithelial cells in tissue culture were treated with proparacaine hydrochloride. At the lowest drug concentration used (0.01 mM), no effects were seen on the actin cytoskeleton. At 1.0 mM, some disruption of stress fibers was evident and actin was redistributed in a diffuse fashion. Many of the intact stress fibers had abnormal morphology, distribution, and orientation. Scanning electron microscopy showed a loss of cell extensions and cell-to-substratum adhesiveness at the leading epithelial edge. Above 1.0 mM, cell spreading was completely abolished and most cells detached from the substratum. After a washout period with drug-free media, these effects were reversible at concentrations of 1.0 mM or less. We postulate that one mechanism by which proparacaine inhibits corneal epithelial migration and adhesion is through alteration of the actin cytoskeleton.