Induction of resistance to aflatoxin B1(AFB1) binding to cellular macromolecules in the rat by chronic exposure to AFB1and aflatoxin M1(AFM1) was investigated. The binding of [14C]AFB1to liver macromolecules was measured in F‐344 rats fed 0.5 ppb or 50 ppb AFM1or 50 ppb AFB1for 41 wk. The animals then received an intragastric dose of [14C]AFB1at 5 μg/kg and were sacrificed 6 h later. Hepatic DNA, RNA, and protein were isolated by chloroform‐phenol extraction and hydroxylapatite chromatography. In animals preexposed to 50 ppb AFB1, labeled AFB1binding to DNA, RNA, and protein was decreased by 72%, 74%, and 61%, respectively. Preexposure to AFM1resulted in a small reduction in binding to nucleic acids. Glutathione trans‐ferase activity was increased by 133% in animals fed 50 ppb AFB1, by 48% in those preexposed to 50 ppb AFM1, and remained at control values in rats fed 0.5 ppb AFM1. These results suggest that the induction of detoxification enzymes following chronic exposure to aflatoxin might contribute to the reduction in covalent binding of AFB1to macromolecules.