The tight junction is composed of many proteins and includes three members of the MAGUK (membrane-associated, guanylate kinase-like) protein family: ZO-1, ZO-2, and ZO-3. ZO-2 was cloned and sequenced from embryonic chicken retina. Antibodies against a short ZO-2 peptide immunolabeled the outer limiting membrane (an adherens junction of the neural retina) and the apical junctional complexes of the retinal pigment epithelium. Each ZO family member contains a homologous series of protein-binding domains: three distinct PDZ domains and src homology 3 (SH3), guanylate kinase-like (GuK), and acidic domains. Compared with human and canine ZO-2s, the PDZ and SH3 domains are the most conserved (90-95% amino acid sequence identity). These domains are only 50-71% identical with the homologous domains of ZO-1 and ZO-3. Although the sequence is less conserved for regions that link the protein-binding domains, the length of those regions is conserved in ZO-2s. The postacidic (C-terminal) region is the least conserved. The evolutionary pressure to maintain the sequence of the protein-binding domains suggests that homologous domains have different functions in ZO-1, ZO-2, and ZO-3.