Aflatoxin B₁ (AFB₁) is a highly hepatotoxic and carcinogenic mycotoxin produced by Aspergillus species. The compound is mainly metabolized in the liver and its metabolism varies between species. The present study quantified relevant AFB₁- metabolites formed by mouse, rat, and human primary hepatocytes after treatment with 1 µM and 10 µM AFB₁. The use of liquid chromatographic separation coupled with tandem mass spectrometric detection enabled the selective and sensitive determination of phase I and phase II metabolites of AFB₁ over incubation times of up to 24 h. The binding of AFB₁ to macromolecules was also considered. The fastest metabolism of AFB₁ was observed in mouse hepatocytes which formed aflatoxin P₁ as a major metabolite and also its glucuronidated form, while AFP₁ occurred only in traces in the other species. Aflatoxin M₁ was formed in all species and was, together with aflatoxin Q₁ and aflatoxicol, the main metabolite in human cells. Effective epoxidation led to high amounts of DNA adducts already 30 min post-treatment, especially in rat hepatocytes. Lower levels of DNA adducts and fast DNA repair were found in mouse hepatocytes. Also, protein adducts arising from reactive intermediates were formed rapidly in all three species. Detoxification via glutathione conjugation and subsequent formation of the N-acetylcysteine derivative appeared to be similar in mice and in rats and strongly differed from human hepatocytes which did not form these metabolites at all. The use of qualitative reference material of a multitude of metabolites and the comparison of hepatocyte metabolism in three species using advanced methods enabled considerations on toxification and detoxification mechanisms of AFB₁. In addition to glutathione conjugation, phase I metabolism is strongly involved in the detoxification of AFB₁.