Recent research suggests that tight junctions (TJs) are located in the stratum granulosum, where they contribute to the barrier function of the epidermis. In this study, we investigated the formation of functional TJs in cultured normal human epidermal keratinocytes. We observed the development of permeability barrier function through the process of Ca(2+)-induced differentiation. Immunofluorescence analyses at 96 h after Ca(2+)-induced differentiation revealed concentrated portions of occludin, a TJ-specific marker, arranged as continuous lines circumscribing individual flattened suprabasal cells in areas with high concentrations of claudin-1 and -4. Transient Ca(2+) depletion reversibly disrupted the continuous network of TJ proteins and the permeability barrier. We also found that the addition of ochratoxin A weakened the permeability barrier and the expression of claudin-4. Our findings suggest that TJ proteins contribute to the permeability barrier in epidermal keratinocytes.