The metabolism of [14C]aflatoxin B1 by 9000 X g supernatant fraction of livers of duck, rat, mouse, monkey, and humans was compared by incubating the compound and liver fractions in the presence of cofactors and air for 30 min. The incubation medium was extracted with chloroform, and the soluble metabolites were separated on thinlayer plates and quantified; radioactivity remaining in the aqueous phase was determined in order to quantify metabolism to water-soluble derivatives. Duck, monkey, and human livers were most active in total conversion, each metabolizing approximately 80% of available substrate in 30 min. Rat and mouse livers had lower activites, metabolizing 15 to 20%. Duck liver produced mainly (60%) chloroform-insoluble derivatives, but all other species produced larger quantities of chloroform-soluble than insoluble metabolites. Aflatoxin Q1 was the principle chloroform-soluble metabolite produced by monkey, human, and rat liver, whereas duck liver produced mainly aflatoxicol in that fraction. Aflatoxin P1 was produced by monkey, human, and mouse liver, but not by duck and rat. The chromatographic region containing M1 and B2alpha contained low levels of radioactivity in all species except human. No consistent pattern of metabolism emerged which could be correlated with species differences in response to aflatoxin B1 toxicity or carcinogenicity.