The corneal epithelium, like other stratifying epithelium, does not present a well formed junctional complex as compared to that of simple epithelia. However, the resistance barrier of the corneal epithelium is to a great extent generated by zonula occludens (ZO, tight junction), which are formed between the cells of the apical-most strata. The tight junction provides a continuous seal around the apical aspect of adjoining epithelial cells, thereby preventing the free passage of molecules between adjacent epithelial cells (paracellular pathway). We have examined rabbit corneal epithelia with monoclonal antibody against the tight junction associated protein ZO1. With this antibody, we resolved two distinct patterns of ZO1 expression, one being the lateral boundary of the apical cell, which appeared as a true zonula around these cells. The second pattern of expression for ZO1 was at a set of punctate spots that correspond to the connection of the most apical portion of the basal corneal epithelial cells, with the above wing cells. En face, confocal analyses revealed that these areas consisted of 5-6 distinct spots per basal cell at or near the contact points with the immediate wing cells above. ImmunoEM revealed that the mid-epithelial accumulations of ZO1 were not tight junctions, but rather a form of adherens junction. The expression of ZO1 in the mid-epithelial level of the cornea is neither correlated with the presence of tight junction, nor with the established barrier functions. Interestingly, these junctions in the corneal epithelium also contain paxillin, a focal adhesion associated phosphoprotein which is a target of pp125 focal adhesion kinase, erbB-2 kinase and p21Obcr/abl oncogene. We postulated that the ZO1/paxillin adherens junction within stratified epithelium, such as the corneal epithelium, may function to reinforce attachments at the level of the basal cell to wing cell junction and be regulated by reversible phosphorylation. We speculate that the regulated phosphorylation of tyrosine residues on paxillin may perform a critical role in controlling epithelial cell-cell interactions as it does in cell-matrix adhesion.