Primary mouse hepatocyte cultures are widely used in toxicological and pharmacological studies. However, the strain differences in alterations of metabolic enzymes and the regulation of gene expression in response to different stimuli remains unclear. To address this issue, we examined the expression of metabolic enzymes and the regulatory role of DNA methylation in the primary hepatocytes of two mouse strains, CD-1 and C57BL/6. Primary culture of mouse hepatocytes was established using collagen sandwich configuration. Analysis of gene expression of 24 phase I, 18 phase II, and 6 phase III metabolic enzymes on 4 consecutive days after cell seeding revealed that the basal levels of most enzymes in primary cultured hepatocytes differed greatly between the two mouse strains. However, the dynamic changes in most genes were identical between the two strains. In addition, treatment with 3-methylcholanthrene, phenobarbital, and rifampin led to the induction of cytochrome P-450 (cyp) 1a1 and cyp1a2, cyp2b10, cyp3a11. However, induction varied in degree between the two types of primary hepatocytes. The dynamic changes in global DNA methylation and the expression of DNA methylation regulatory factors of the two mouse strains were similar. Of the genes down-regulated over the culture period, hypermethylation of cyp2e1 gene appeared in both mouse strains and led to a suppression of gene expression. Taken together, these results demonstrate that the expression of metabolic enzymes and the response to agonists in primary hepatocytes differ between CD-1 and C57BL/6 mouse strains. Epigenetic regulation might be involved in the suppression of cyp 450s' expression.