Animal models have been used to investigate the involvement of epithelial tight junctions in the pathogenesis of human airway disease. However, no previous study has compared the tight junction morphologies of human and animal species in order to relate findings in animal models to human disease. In the present study, we therefore undertook a comprehensive quantitative evaluation of tight junction morphology, to determine what similarities or differences may exist in rat and human airways. Human tissue was obtained from grossly and histologically normal extrapulmonary bronchi from lungs resected for pulmonary tumour (n = 8); rat tracheal epithelium was acquired from Sprague-Dawley specific pathogen-free animals (n = 12). The tight junction morphologies of the two species were compared with respect to junctional depth, number of strands and junctional complexity. The basic architectural arrangement of the tight junctions in both species was found to be similar; however, tight junctions in rat tissues were less deep, comprised fewer strands, and had fewer strand interconnections compared with those in the human samples. The number of strands per interconnection was similar in the two species. We conclude that, in spite of a general similarity of rat and human airway epithelial tight junctions, there are specific quantitative details of morphology which need to be considered when attempting to extrapolate to the human the results of studies of airway epithelial permeability conducted in the rat. The precise biological significance of these differences, as yet, remains unclear.