Enzymic depletion of glutathione (GSH) in vitro by aniline analogs was mostly dependent on the cytochrome P-450 level in liver microsomes. In a case of acetaminophen (AAP), active metabolite of AAP formed through liver microsomal drug metabolizing enzymes consumed GSH. The active metabolite formed binds, at least in part, covalently to liver microsomal proteins. In addition, species differences in the extent of GSH depletion by AAP in vitro was related to the amounts of the active metabolite of AAP bound covalently to liver microsomal protein(s) by experiments using 14C-AAP. Similar depletion of GSH was also seen with other aniline analogs such as aniline itself and p-chloroaniline, but not with acetanilide, in four animal species. These in vitro results obtained here strongly support the well-known findings concerning both GSH depletion and covalent binding in vivo of the active metabolite after AAP treatment.