This study demonstrates the presence of tight junction antigens in adult and developing human epidermis. Indirect immunofluorescence labeling and immunoelectron microscopy with antibodies to ZO-1 and occludin localized tight junction components ZO-1 and occludin to a narrow zone of the granular cells of adult epidermis. Double immunolabeling for tight junction components with adherens junction or desmosome proteins suggested that occludin is more specific for tight junctions than ZO-1, which may also be associated with adherens junctions. In developing skin, tight junctions interconnected the peridermal cells, and after the fetal stratification localized to the granular cell layer. Immunolabeling of psoriasis, lichen planus, and ichthyosis vulgaris, representing aberrant differentiation of the epidermis, showed that these conditions were associated with relocation of ZO-1 and occludin to the spinous cells. Cultures of epidermal keratinocytes, which offer a useful model for the formation of cellular contacts, revealed that tight junction components, ZO-1 and occludin, displayed a marked degree of colocalization relatively late during the process when the fusion zone had assumed a linear appearance. This suggests that the formation of adherens junctions and desmosomes precedes that of tight junctions. We speculate that the epidermal barrier, isolating the human body from the external environment, is in part formed by tight junctions of stratum granulosum.