A strain specificity has been demonstrated for the effect of subsequent administration of phenobarbital, in which diethylnitrosamine-initiated hepatocarcinogenesis was promoted in C3H mice, inhibited in B6C3F1 (C57BL X C3H) and not affected in C57BL mice. Therefore, the study examined in these three strains of mice and in C3B6F1 (C3H X C57BL) mice the effect of phenobarbital administered in their drinking water for 4 days or 28 days. The liver weight to body weight ratio was increased by phenobarbital in all types of mice. Microsomal protein concentrations were increased in C57BL mice after 28 days of treatment, in C3H after both 4 days and 28 days and in B6C3F1 after 4 days of treatment. No effect upon microsomal protein content was observed in C3B6F1 mice. DNA content was increased in C3H mice, both in 4 days and 28 days of treatment groups, while the other strains showed either a decrease or no difference from control. DNA synthesis was elevated in all strains of mice after 4 days of treatment with phenobarbital, however, after 28 days of treatment there was either a much reduced increase (C57B1 and C3B6F1) or no difference (C3H and B6C3F1) from controls. In all four types of mice after 4 and 28 days of treatment, phenobarbital increased the concentration of cytochrome P-450, the activity of aminopyrine-N-demethylase and 7-ethoxyresorufin-O-deethylase and the oxidation of testosterone. The oxidative metabolites of testosterone were similar in the four types of mice.