Female Sprague-Dawley rats were given a single dose of tamoxifen (20 mg/kg body weight) by gavage or the same dose at 24-hr intervals for 2, 12, or 52 weeks, and the altered expression of cyclin-dependent kinase-interacting protein p21 (p21), tumor suppression protein p53, and the placental form of glutathione S-transferase (GST-P) in the liver was comparatively examined during the process of tamoxifen-induced hepatocarcinogenesis. The development of hepatocellular carcinoma was histopathologically observed only in rats administered tamoxifen for 52 weeks, but not in any other experimental groups. Significant increases in levels of the mRNA and protein of p21 were first observed in rats administered tamoxifen for 2 weeks, and the levels increased with further long-term treatment. Immunohistochemical analyses of p21 and GST-P in the liver of rats administered tamoxifen for 12 and 52 weeks revealed that the localization of p21-positive cells did not necessarily coincide with that of GST-P-positive cells. In the 52-week group, p21-positive cells, rather than GST-P-positive cells, were observed preferentially in the region consisting of histopathologically malignant cells. The present study demonstrated that during the process of tamoxifen-induced hepatocarcinogenesis, expression levels of the mRNA and protein of p21 were increased. The significance of the increased expression of p21 during hepatocarcinogenesis is discussed.