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The mammalian protein ASIP/PAR-3 interacts with atypical protein kinase C isotypes (aPKC) and shows overall sequence similarity to the invertebrate proteins C. elegans PAR-3 and Drosophila Bazooka, which are crucial for the establishment of polarity in various cells. The physical interaction between ASIP/PAR-3 and aPKC is also conserved in C. elegans PAR-3 and PKC-3 and in Drosophila Bazooka and DaPKC. In mammals, ASIP/PAR-3 colocalizes with aPKC and concentrates at the tight junctions of epithelial cells, but the biological meaning of ASIP/PAR-3 in tight junctions remains to be clarified. In the present study, we show that ASIP/PAR-3 staining distributes to the subapical domain of epithelial cell-cell junctions, including epithelial cells with less-developed tight junctions, in clear contrast with ZO-1, another tight-junction-associated protein, the staining of which is stronger in cells with well-developed tight junctions. Consistently, immunogold electron microscopy revealed that ASIP/PAR-3 concentrates at the apical edge of tight junctions, whereas ZO-1 distributes alongside tight junctions. To clarify the meaning of this characteristic localization of ASIP, we analyzed the effects of overexpressed ASIP/PAR-3 on tight junction formation in cultured epithelial MDCK cells. The induced overexpression of ASIP/PAR-3, but not its deletion mutant lacking the aPKC-binding sequence, promotes cell-cell contact-induced tight junction formation in MDCK cells when evaluated on the basis of transepithelial electrical resistance and occludin insolubilization. The significance of the aPKC-binding sequence in tight junction formation is also supported by the finding that the conserved PKC-phosphorylation site within this sequence, ASIP-Ser827, is phosphorylated at the most apical tip of cell-cell contacts during the initial phase of tight junction formation in MDCK cells. Together, our present data suggest that ASIP/PAR-3 regulates epithelial tight junction formation positively through interaction with aPKC.