This study identified a novel integral membrane protein, occludin, localized at tight junctions (TJs) in both epithelial and endothelial cells. Using a junctional fraction from chick liver, three monoclonal antibodies (mAbs) specific for a ~65-kD protein were obtained. These mAbs localized the antigen exclusively at TJ, both at the light and electron microscopic levels. Cloning and sequencing of the cDNA encoding this antigen revealed a 504-amino acid polypeptide with a predicted molecular mass of 55.9 kD. The primary structure of occludin contained four putative membrane-spanning segments, similar to connexin, another integral membrane protein in gap junctions. This study provides the first molecular characterization of an integral membrane protein at TJ, opening new avenues for investigating the molecular mechanisms of TJ formation and function.This study identified a novel integral membrane protein, occludin, localized at tight junctions (TJs) in both epithelial and endothelial cells. Using a junctional fraction from chick liver, three monoclonal antibodies (mAbs) specific for a ~65-kD protein were obtained. These mAbs localized the antigen exclusively at TJ, both at the light and electron microscopic levels. Cloning and sequencing of the cDNA encoding this antigen revealed a 504-amino acid polypeptide with a predicted molecular mass of 55.9 kD. The primary structure of occludin contained four putative membrane-spanning segments, similar to connexin, another integral membrane protein in gap junctions. This study provides the first molecular characterization of an integral membrane protein at TJ, opening new avenues for investigating the molecular mechanisms of TJ formation and function.