Zileuton, an inhibitor of 5-lipooxygenase, the initial enzyme in the leukotriene pathway, was marketed as a new treatment for asthma. This drug has been associated with liver toxicity, which has limited its clinical usefulness. We provide evidence here that the liver toxicity likely involves a sequence of biotransformations leading to 2-acetylbenzothiophene (2-ABT), which is subsequently metabolized to give a reactive intermediate(s). In vitro experiments with the human lymphoblast MCL5 cell line demonstrated that 2-ABT is cytotoxic in a P450-dependent manner. Human liver microsome (HLM) incubations with 2-ABT revealed the formation of two short-lived oxidized species, "M + 16" and "M + 32". Both of these metabolites formed adducts in the presence of GSH or NAC. Singly oxidized M + 16 adducts, from either GSH or NAC, appeared to be unstable in acidic medium and eliminated water readily to form a new compound. Authentic synthetic standards demonstrated that 2-ABT-S-oxide M1 corresponded to the M + 16 metabolite and that the S-oxide underwent nucleophilic addition with GSH and NAC to produce the singly oxidized adducts observed in HLM. The S-oxide adducts readily eliminated water to form a rearomatized 2-ABT-GSH adduct or 2-ABT-NAC adduct. Coelution experiments with the synthetic standard confirmed the structure of the eliminated 2-ABT-NAC adduct C1. LC/MS analyses of urine samples collected from rats dosed with zileuton indicate that C1 is a metabolite of zileuton formed in vivo. The in vitro and in vivo data presented here demonstrate the formation of 2-ABT from zileuton and its further bioactivation to a potentially toxic metabolite.