ZO-1 is a 225-kDa peripheral membrane protein present in all tight junctions. It was recently shown to consist of two isoforms that differ in the presence of an internal 80-amino acid domain termed motif-alpha. To obtain information on their distribution and potential functional significance we have localized the two isoforms in rat kidney by using antibodies that recognize either both ZO-1 isoforms or the larger, motif-alpha-containing isoform. By immunofluorescence, staining with both antibodies was demonstrated at all tight junctions of tubular epithelial cells and the epithelial cells of Bowman's capsule. In contrast, the motif-alpha-containing isoform was absent from the slit diaphragms of the glomerular epithelium and the tight junctions of glomerular and peritubular capillary endothelial cells. This restricted isoform expression was confirmed by immunoblot analysis comparing proteins from purified glomeruli with those from kidney cortex or medulla. Thus, while both isoforms are expressed in typical epithelial tight junctions, only a single isoform, lacking motif-alpha, is expressed in the highly specialized slit diaphragms, where the intercellular spaces are normally open, and in endothelial junctions, which are readily opened by physiologic signals. The differential expression of ZO-1 isoforms in structurally and functionally distinct junctions in the kidney suggests that they may contribute to defining the variable functional properties, in particular the lability of these intercellular junctions.