To elucidate what changes in the expression of gap junction proteins (connexins) occur at what stages during multistage mouse skin carcinogenesis in vivo, we immunohistochemically and morphometrically analyzed the expression of connexin 26 (C×26) and connexin 43 (C×43) in papillomas, well-, moderately- and poorly-differentiated squamous cell carcinomas, as well as in squamous cell carcinomas at invasion sites and those metastasized into lymph nodes in female CD-I mice as a result of treatment with dimethylbenz[a]anthracene and 12-O-tetradecanoylphorbol-13-acetate. In papillomas, no clear reduction of the two connexins was observed; however, C×26 and C×43 were frequently co-localized in the same gap junction plaques, whereas the two kinds of C×s were differentially expressed in normal and surrounding non-tumorous epidermis. In squamous cell carcinomas, the expression of both C×26 and C×43 significantly decreased compared with surrounding non-tumorous epidermis and papillomas. The Western blot analysis confirmed that both C×26 and C×43 proteins were reduced in squamous cell carcinomas compared with papillomas. Furthermore, the expression of C×26 was reduced as cancer cells became morphologically less differentiated, while that of C×43 did not change. Squamous cell carcinomas at invasive sites showed clear reduction of C×26 and C×43. In squamous cell carcinomas metastasized into lymph nodes, C×26 was expressed, but few carcinoma cells expressed C×43. The localization of E-cadherin on the plasma membrane between cancer cells was maintained even at invasive and metastatic sites. Our data suggest that quantitative and qualitative changes in connexin expression are associated with tumor progression, including the loss of differentiation, and invasion and metastasis, during multistage mouse skin carcinogenesis.