The effect of phenobarbital (PB) pretreatment on the metabolism, covalent binding, and cytotoxicity of [14C]aflatoxin B1(AFB1) was studied in primary hepatocyte cultures. Hepatocytes from control and PB‐pretreated rats were isolated from perfused liver biopsies and cultured in a chemically defined, hormone‐supplemented medium. [14C]AFB1, dissolved in medium, was added to cultures at 20–22 h. The metabolism of AFB1 to water‐soluble products and its binding to trichloroacetic acid‐precipitable macromolecules were assessed 0.5 to 24 h later. At 6 h, PB pretreatment reduced total binding to macromolecules by 31% and reduced binding to RNA and DNA by 61% and 66%, respectively. In addition, PB protected cultures from the cytotoxic effects of AFB1, as evidenced by a significantly reduced (p <0.05) leakage of lactate dehydrogenase into the medium at 51 h. Elevated mixed‐function oxidase and giutathione S‐transferase activities, as well as higher levels of AFB1‐giutathione conjugate were measured in cultures from rats pretreated with PB. The protective action of PB was concluded to be due to the induction of hepatic giutathione S‐transferases responsible for the detoxification of A FB1.