Hepatic levels of S-adenosylmethionine (AdoMet), of glutathione, and of the microsomal enzymes p-nitroanisole demethylase and benzo(a)pyrene hydroxylase were measured in male and female rats fed a diet marginally deficient in choline and methionine and void of folic acid (lipotrope deficient) or an adequate diet for 0 to 14 weeks with and without added 2-acetylaminofluorene (AAF). The urinary metabolites of AAF were determined throughout the experimental period. After 2 to 4 weeks of dietary administration, the hepatic AdoMet levels were 43% lower in male rats fed the lipotrope-deficient diet than in male rats fed the lipotrope-adequate diet; no differences were found in hepatic AdoMet of females fed the lipotrope-deficient or lipotrope-adequate diets for 2 to 14 weeks. Administration of AAF to lipotrope-deficient female rats for 2 weeks led to a transient decrease in hepatic levels of AdoMet. The administration of AAF for 2 to 14 weeks did not significantly affect hepatic AdoMet in female rats fed the lipotrope-adequate diet or in male rats fed either diet. Female rats fed the lipotrope-deficient diet and treated with AAF excreted decreased proportions of N-hydroxy-2-acetylaminofluorene and increased proportions of 5-hydroxy-2-acetylaminofluorene in their urine. However, the urine of lipotrope-deficient male rats treated with AAF contained increased proportions of N-hydroxy-2-acetylaminofluorene and decreased levels of 5-hydroxy-2-acetylaminofluorene. The urinary excretion of 7-hydroxy-2-acetylaminofluorene by male and female lipotrope-deficient rats treated with AAF was generally similar to that in lipotrope-adequate rats. The lipotrope-deficient diet did not appear to alter the hepatic levels of glutathione, p-nitroanisole demethylase, or benzo(a)pyrene hydroxylase activity was lower in the livers of lipotrope-deficient male rats treated with AAF for 8 to 14 weeks than in the livers of lipotrope-deficient rats not receiving the carcinogen. The altered metabolism of AAF correlated well with the previously reported effects of a marginal lipotrope deficiency on AAF carcinogenesis.