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The goal of the present study was to determine the rate of restoration of the corneal epithelial barrier following a superficial keratectomy using a functional assay of tight junction integrity. Adult Sprague–Dawley rats were anesthetized and a 3-mm superficial keratectomy was performed. The eyes were allowed to heal from 4 h to 8 weeks and the rate of epithelial wound closure was determined. To examine the restoration of the barrier function, EZ-Link Sulfo-NHS-LC-Biotin (LC-Biotin) was applied to all eyes, experimental and control, for 15 min at the time of sacrifice. This compound does not penetrate through intact tight junctions. Indirect immunofluorescence was performed with anti-laminin, a marker of basement membrane; fluorescein-conjugated streptavidin to detect the biotinylated marker; and anti-occludin and anti-ZO-1, markers of tight junctions. Epithelial wound closure was observed at 36–42 h after wounding. LC-Biotin did not penetrate the intact epithelium. Upon wounding, LC-Biotin penetrated into the stroma subjacent and slightly peripheral to the wound area. This pattern was present from 4–48 h post-wounding. The area of LC-Biotin localization decreased with time and the functional barrier was restored by 72 h. Occludin and ZO-1 were present at all time points. The number of cell layers expressing these proteins appeared to increase at 48 and 72 h. Continuous laminin localization was not observed until at least 7 days after wounding. Barrier function is restored within 1–1.5 days after epithelial wound closure. The loss of barrier function does not extend beyond the edge of the original wound. The restoration of barrier function does not appear to correlate with reassembly of the basement membrane in this model.