Background/Aims: The synthetic estrogen 17 alpha-ethinyl estradiol (EE), a potent tumor promoter in rat liver, stimulates growth during short-term treatment but inhibits hepatocyte proliferation upon prolonged treatment. To identify the molecular targets of the mitoinhibitory effect of EE, the expression of proteins regulating G(1)- and S-progression were analyzed during the first cell cycle in EE-treated female Wistar rats. Methods: Long-term (60 days) EE treatment. Immunohistochemical staining for proliferation cell nuclear antigen (PCNA) to detect cells in S phase and quantification of mitosis. Western blot to monitor protein expression. Cdk2 kinase assay to examine histone H1 phosphorylation. Results: EE reduced the number of cells in S phase and mitosis by about 70%. Cyclin D-1 and D-3 were unaffected, while cdk4 was moderately decreased. Cyclin E and cdk2 were markedly decreased with concomitant marked reduction of cdk2 kinase activity. EE also decreased cyclin A and increased G(1) levels of p53 and p21. Conclusions: EE causes a cell cycle block before S-phase. The reduction of the cdk2 kinase activity, essential for G(1)/S-transition, might be involved in the cell cycle block. Also, EE treatment results in p53 activation and upregulation of the cdk inhibitor p21 that might contribute to the G(1) arrest.