The tight junctions (TJs) present exclusively in between the superficial cells (SC) are an important component for the barrier and ion secretory functions of the corneal epithelium. Electrophysiological studies have demonstrated that digitonin-induced devitalization of overlying cells induces the de novo generation of a paracellular barrier between the intrastratal cells becoming exposed to the outer surface. It was also shown that the closer the exposed cells to the SC position the higher their competency for this activity. We have now examined the spatial and quantitative distribution of ZO-1, a protein closely associated with the cytosolic face of TJs in the rabbit. Immunohistology showed: (a) no detectable ZO-1 in basal cells; (b) incipient punctuate accumulations in the wing cells; (c) numerous foci and a diffuse cytosolic staining in the squamous (SQ) cells; and (d) strong staining at the apical TJ locations in the superficial SQ layer. Immunoblot analysis of separated layers showed a SQ:basal cell ZO-1 concentration ratio in excess of 100. Devitalization of the SQ layers induced strong ZO-1 synthesis in the newly exposed wing cells where electron microscopy showed time-dependent development of TJs. A similar ZO-1 increase was observed in the basal cells after removal of all suprabasal cells by a low [Ca2+] incubation method. These results provide a biochemical correlate to the previous electrophysiological findings and show that expression of ZO-1 in the corneal epithelium is intimately related to the development of the superficial cell phenotype.