The effects of phenobarbital (PB) and other liver monooxygenase modifiers on dimethylnitrosamine (DMN)-induced alkylation of rat liver DNA and protein were investigated at different carcinogen doses. In rats given single injections of radioactively labeled DMN, pretreatment with PB (80 mg/kg body weight, administered for 5 days) resulted in a small but significant decrease in the formation of 7-methylguanine and O6-methylguanine per mole of guanine in liver DNA associated with a decrease in the O6/N7-methylguanine ratio. The specific radioactivity of liver protein was also lowered in PB-pretreated rats. The degree of PB interference was independent of DMN dose within a carcinogen dose range of 0.5 microgram to 10 mg/kg body weight. In parallel experiments, the effects of pretreatment with PB, Aroclor 1254, pregnenolone-16 alpha-carbonitrile, butylated hydroxytoluene, beta-naphthoflavone, and ethanol on DMN-induced alkylation of liver DNA were studied at a DMN dose of 5 micrograms/kg body weight. In general, pretreatment with these modifiers of liver monooxygenase resulted in a decrease in specific alkylation of DNA and in the ratio of 7-methylguanine to guanine. If, however, 7-methylguanine levels were related to total liver DNA, these differences in DNA alkylation between controls and pretreated rats became substantially smaller, partially being negligible, since these inducers led to an increase in relative liver weight with concomitant increase in the content of liver DNA. Thus, when expressed per total liver, no significant changes in the overall extent of metabolic activation of DMN were evident. These findings are not consistent with the results of in vitro studies on DMN metabolism in microsomal systems which favored the hypothesis that changes in the metabolism of hepatocarcinogens are responsible for the reduction of liver tumor response in animals treated simultaneously with inducers of the liver monooxygenase system and hepatocarcinogens. Our findings suggest that these effects might rather be related to drug-mediated changes on the cellular level.