The administration of 3'-hydroxyacetanilide, a regioisomer of acetaminophen, to mice failed to produce hepatotoxicity even after the administration of diethyl maleate. In contrast, hepatotoxicity did occur when 3'-hydroxyacetanilide was administered to buthionine sulfoximine pretreated mice. Although the administration of 3'-hydroxyacetanilide in conjunction with either diethyl maleate or buthionine sulfoximine depleted total hepatic glutathione, only the combined buthionine sulfoximine-3'-hydroxyacetanilide treatment decreased hepatic mitochondrial glutathione concentrations to below 20% of control values. In addition, pretreatment with buthionine sulfoximine increased the amount of 3'-hydroxyacetanilide bound to mitochondrial proteins. These results, in conjunction without previous results on the involvement of mitochondrial damage in the pathogenesis of hepatotoxicity caused by acetaminophen, suggest a probable relationship between mitochondrial damage caused by the buthionine sulfoximine-3'-hydroxyacetanilide treatment and hepatotoxicity caused by this treatment.