OBJECTIVE The authors examined the effect of thrombin on confluent bovine corneal endothelial (BCE) cells in vitro, especially on cellular integrity and on the redistribution of F-actin and vinculin. METHODS Immunofluorescent stainings against F-actin and vinculin were carried out on confluent BCE cells, and the effect of thrombin was evaluated. RESULTS F-actin was distributed at the cytoplasmic peripheries of confluent BCE cells, forming dense peripheral bands (DPB), whereas vinculin was linearly located at the cell borders. Enzymatically active thrombin caused the loss of DPB and an increase of central microfilament bundles, associating the dissociation of vinculin-cell plaques and the formation of intercellular gaps. However, enzymatically inactive thrombin did not induce such changes. The thrombin effect was reversible and occurred in a concentration-dependent manner. The pre-incubation of BCE cells with disrupting agents of microtubules, such as colchicine and demecolcine, or voltage-dependent Ca2+ channel blockers did not affect these thrombin-induced changes, whereas forskolin and energy blockers such as oligomycin AB and C and antimycin A inhibited these changes. CONCLUSIONS Enzymatic-active thrombin affects the arrangement of the cytoskeletal structure of BCE cells and cell-substratum interaction and plays an important role in the re-integrity or repair processes of the monolayer of BCE cells.