Preservatives are a major component of the ophthalmic preparations in multi-dose bottles. The purpose of this study was to investigate the acute effect of benzalkonium chloride (BAC), a common preservative used in ophthalmic preparations, on the localization and expression of zonula occludens-1 inthe rabbit corneal epithelium in vivo. BAC at 0.005%, 0.01%, or 0.02% was topically applied to one eye each of albino rabbits at 5 min intervals for a total of 3 times. The contralateral untreated eyes served as controls. The following clinical indications were evaluated: Schirmer test, tear break-up time (BUT), fluorescein and roseBengalstaining. The structure of central cornea was examined by in vivo confocal microscopy, and the corneal barrier function was evaluated by measurement of corneal transepithelial electrical resistance (TER). Whole mount corneas were analyzed by using fluorescence confocal microscopy for the presence of zonula occludens (ZO)-1, 2, occludin, claudins, Ki67 and cell apoptosis in the epithelium. The expression of ZO-1 inthe corneal epithelium was also examined by western blot and reverse transcription-polymerase chain reaction analyses. Exposure to BAC resulted in higher roseBengalstaining scores while no significant changes in BUT, Schirmer and corneal florescein scores. It also induced corneal epithelial cell damage, dispersion of tight junction proteins from its normal locus at the superficial layer and declines in corneal TER. However, the amounts of ZO-1 mRNA and protein in the corneal epithelium were not reduced by BAC treatment. Exposure to BAC can quickly impair the corneal epithelium without tear deficiency. BAC disrupts the tight junctions of corneal epithelium between superficial cells in the rabbit corneal epithelium in vivo.