DNA methylation is an important epigenetic mechanism involved in transcriptional control and altered patterns of methylation may lead to the aberrant gene expression contributing to carcinogenesis. Three groups of mice were used in the current study: the relatively liver-tumor-sensitive C3H/He strain and B6C3F1 stock (C57BL/6xC3H/He), as well as the relatively resistant C57BL/6 strain. For a 2-week period, animals from each group were given drinking water containing a tumor-promoting dose of phenobarbital (PB), a nongenotoxic rodent carcinogen. Methylation-sensitive restriction digests using HpaII or MspI were followed by PCR amplification using an arbitrary primer or primer pair, binding preferentially to guanine and cytysine (GC)-rich regions of DNA, including CpG islands. This procedure allows for assessment of methylation at the internal and/or external cytosine of the 5'-CCGG-3' sites recognized by MspI and HpaII. Results with the single primer indicated marked differences in PB-induced hypermethylation at external and internal cytosines of 5'-CCGG-3' sites: C3H/He >> B6C3F1 > C57BL/6. Results with the arbitrary primer pair indicated PB-induced hypermethylation at the external cytosine of 5'-CCGG-3' site: B6C3F1 > C3H/He, and a low level of hypomethylation at internal and external cytosine sites in C57BL/6. Thus, there was a clear indication of more methylation changes in GC-rich regions of DNA, primarily hypermethylation, in the tumor-sensitive groups of mice in response to PB treatment. Therefore, this study supports our hypothesis that the capacity to maintain normal methylation patterns is related inversely to tumor susceptibility.