Tamoxifen is a non-steroidal anti-estrogen used for the treatment of breast cancer and, more recently, as a chemopreventive agent in healthy women at high risk of developing breast cancer. On the other hand, tamoxifen is a potent hepatocarcinogen in rats, with both tumor-initiating and tumor-promoting properties. There is substantial evidence that hepatic tumors in rats are initiated as a result of formation of tamoxifen-DNA adducts; however, events subsequent to DNA adduct formation are not clear. Recently, it has been demonstrated that genotoxic carcinogens, in addition to exerting genotoxic effects, often cause epigenetic alterations. In the current study, we investigated whether or not the mechanism of tamoxifen-induced hepatocarcinogenesis includes both genotoxic and epigenetic components. Female Fisher 344 rats were fed a 420 p.p.m. tamoxifen diet for 6, 12, 18 or 24 weeks. Hepatic tamoxifen-DNA adduct levels, as assessed by high-performance liquid chromatography and electrospray tandem mass spectrometry, were 580 adducts/10(8) nt at 6 weeks, and increased to approximately 1700 adducts/10(8) nt by 18 weeks. Global liver DNA hypomethylation, as determined by an HpaII-based cytosine extension assay, was increased at all time points, with the maximum increase (approximately 200%) occurring at 6 weeks. Protein expressions of maintenance (DNMT1) DNA methyltransferase and de novo DNA methyltransferases DNMT3a and DNMT3b were decreased at all time points. Likewise, trimethylation of histone H4 lysine 20 was significantly decreased at all time points. In contrast, non-target tissues (i.e. mammary gland, pancreas and spleen) did not show any changes in global DNA methylation or DNA methyltransferase activity. These data indicate the importance of genotoxic and epigenetic alterations in the etiology of tamoxifen-induced hepatocarcinogenesis.