Susceptibility to acetaminophen-induced hepatotoxicity was found to vary widely in an outbred colony of Swiss Webster mice. Some acetaminophen-treated male mice showed a significant elevation in serum levels of the hepatic enzyme alanine aminotransferase at a normally non-hepatotoxic oral dose. A selective breeding program over 17 generations produced inbred mice which were either susceptible or nonsusceptible to the hepatotoxic effects of acetaminophen. Liver microsomes from the susceptible group showed a statistically significant increase in the ability to metabolize acetaminophen to a reactive intermediate which covalently binds N-acetylcysteine. Microsomal cytochrome P450 activities associated with CYP1A2 (acetanilide 4-hydroxylation and methoxyresorufin O-demethylase) were significantly increased in the susceptible group. Ethoxyresorufin O-deethylase activity, associated with both CYP1A1 and CYP1A2, was also significantly elevated in this group. Further examination of both CYP1A isoforms revealed that hepatic CYP1A1 and CYP1A2 mRNA and protein levels were significantly elevated in animals from the susceptible group. In vivo caffeine 3-demethylation, which is associated with CYP1A2 activity, co-segregated with acetaminophen susceptibility and showed a significant positive correlation (r = 0.626, p < 0.005) with CYP1A2 mRNA expression in animals from both the susceptible and nonsusceptible groups. The co-segregation of elevated basal Cyp1a1 and CYP1a2 gene expression levels in animals selected for susceptibility to acetaminophen-induced hepatotoxicity suggested a common heritable basis for regulation of basal expression of both of these CYP1A isoforms. This was supported by the correlated expression of both CYP1A mRNAs within individual mice (r = 0.644, p < 0.02).