tert-Butyl-4-hydroxyanisole (BHA) and its demethylated analog, tert-butyl-hydroquinone (TBHQ), are antioxidants used in food. Both BHA and TBHQ have been shown to promote kidney and bladder carcinogenesis in the rat. We have previously demonstrated that glutathione (GSH) conjugates of a variety of hydroquinones are nephrotoxic and proposed that GSH conjugation serves to target these compounds to the kidney. In the present study, we examined the metabolism of TBHQ, focusing on the formation of potentially nephrotoxic sulfur-containing metabolites. 2-tert-Butyl-5-glutathion-S-ylhydroquinone, 2-tert-butyl-6-glutathion-S-ylhydroquinone, and 2-tert-butyl-3,6-bisglutathion-S-ylhydroquinone were identified as biliary metabolites of TBHQ (1.0 mmol/kg, ip) in male F344 rats, accounting for 2.2% of the dose. Liquid chromatography/mass spectroscopic analysis of urine also revealed the presence of additional sulfur-containing metabolites, tentatively identified as 2,5-dihydroxy-3-tert-butyl-thiophenol, 2,5-dihydroxy-4-tert-butylthiophenol, and their S-methyl derivatives. No mercapturic acids of TBHQ were found in the urine. The major biliary and urinary metabolites were TBHQ-glucuronide and TBHQ-sulfate, with a trace of TBHQ excreted unchanged. The results indicate that TBHQ undergoes oxidation and GSH conjugation in vivo in the male F344 rat. These conjugates are excreted into bile and undergo further metabolism prior to excretion in urine. Formation of the S-containing metabolites of TBHQ may occur in amounts sufficient to play a role in the toxicity of TBHQ to kidney and bladder.