Abstract The present article investigates the role of cell cytoskeleton on tight junction permeability in the cornea by combined methods based on microscopy and electrophysiology. Ultrastructural characterization of the cytoskeletal network of the cornea was performed using the S-1 myosin subfragment labeling technique and transmission electron microscopy. The effect of various cytoskeleton-active agents including cytochalasin B, phalloidin, colchicine, kinetin, and a calcium chelator, EDTA on the cytoskeleton and tight junction permeability was also studied using confocal fluorescence microscopy and electrical resistance measurements. Results from this study indicate that actin microfilaments, a major component of the cytoskeletal system, play a major role in controlling tight junction permeability of the cornea. Morphologically, these filaments are present throughout the entire cornea with heavy accumulation near the regions of tight junctions, superficial surfaces, and spot-desmosomes of the intercellular spaces. Treatment with EDTA and cytochalasin B results in a disruption of these filaments and subsequent reduction in electrical resistance. This effect is fully reversible upon removal of the compounds, except for prolonged exposure. Compounds that stabilize these filaments, i.e., phalloidin and kinetin, exhibit a reverse effect on the comeal resistance. Colchicine, a microtubule-selective agent, has no effect on the resistance. The correlation between cytoskeletal patterns and electrical resistance in the presence of drugs suggests that actin microfilaments are associated with the tight junction complex and play an important role in regulating the degree of sealing of the paracellular transport pathway.