Abstract In male and female DDY/Slc mice given single oral doses (20 or 500 mg/kg body weight) of 3,5-di- tert-butyl-4-hydroxytoluene (BHT) labelled with 14C at the p-ethyl group, 14C was distributed mainly in the stomach, intestines, liver and kidney, and then excreted in the urine, faeces and expired air. During the 7 days after treatment, 41–65, 26–50 and 6–9% of the 14C dose was excreted in faeces, urine and expired air, respectively, and the total recovery was 96–98%. Levels of 14C in 21 male and 22 female tissues 7 days after treatment were less than 1 μg BHT equivalents/g tissue (ppm) in mice given 20 mg/kg and less than 11 ppm in mice given 500 mg/kg. When [ 14C]BHT was given orally to male mice at 20 mg/kg/day for 10 days, 14C was rapidly excreted and did not exhibit any tendency to accumulate in any tissues. Thin-layer chromatography and high-performance liquid chromatography analyses showed that more than 43 metabolites were present in the urine and faeces of both species, and all of these were identified to determine metabolic pathways for BHT in mice and rats. Major metabolic reactions of [ 14C]BHT in mice were the oxidation of the p-methyl group attached to the benzene ring and of the tert-butyl groups. The products from the latter reaction were cyclized to some extent by reacting with the adjacent phenolic OH group to give hemiacetals or lactones. The carboxyl derivatives from the p-methyl oxidation were conjugated with glucuronic acid. When single oral doses of 20 or 500 mg [ 14C]BHT/kg were given to male Sprague-Dawley rats, metabolites similar to those in mice were found. However, the major biotransformation was oxidation of the p-methyl group, and oxidation of the tert-butyl groups was a minor reaction in rats.