We investigated the immunohistochemical expression of Rb protein (pRb), which plays an important role in the regulation of the cell cycle, in rat tongue carcinoma induced by 4-nitroquinoline 1-oxide. In addition, we made an immunohistochemical investigation of cyclin D1 and cdk4, which are involved in the Rb pathway. The labeling index of pRb expression in cases with carcinoma was significantly decreased compared with that in cases with a premalignant lesion (P<0.01), while the labeling index of cyclin D1 and cdk4 increased gradually during the course of carcinogenesis. We analyzed the phosphorylation of pRb by immunoblotting using G3-245 monoclonal antibody, which recognizes both the phosphorylated and unphosphorylated forms of pRb. Although expression of the phosphorylated pRb band was notably increased in dysplastic membrane compared with the control membrane, it almost disappeared in cases with carcinoma. Unphosphorylated pRb bands were also expressed in control membrane and dysplastic membrane but not in cases with carcinoma. In conclusion, a decrease of pRb and an increase of cdk4 and cyclin D1 were shown to occur during the premalignant stage. The decrease of pRb in quantity and the increase of its phosphorylation may prevent G1 arrest and consequently accelerate proliferation of the chemically injured cells contributing to the initiation of carcinogenesis.