The teratogenicity of phenytoin may result from its enzymatic bioactivation to a reactive intermediate, which, if not detoxified, can interact with embryonic tissues and alter development. Glutathione (GSH) is an important cofactor/substrate for many physiological processes and for the detoxification of xenobiotic reactive intermediates. This study examined the effects of the GSH depletor diethyl maleate (DEM) and the GSH synthesis inhibitor L-buthionine-(S,R)-sulfoximine (BSO) on phenytoin embryopathy. Phenytoin, 55 mg/kg, was administered intraperitoneally (ip) to pregnant CD-1 mice at 0900 hr on gestational days 12 and 13. Pretreatment with DEM, 150 or 300 mg/kg ip, enhanced the incidence of phenytoin-induced cleft palates by 3.3-fold and 2.3-fold, respectively (P less than 0.05), without affecting the incidence of resorptions, postpartum death, or mean fetal weight. BSO, 1,800 mg/kg ip, given 0.5 hr prior to phenytoin, resulted in a 2.4-fold increase in postpartum lethality and a 5-fold increase in fetal weight loss (P less than 0.05), without altering the incidence of resorptions or cleft palates. In two subsequent studies, BSO, 680-1,018 mg/kg/day, was given in the drinking water on gestational days 9 to 13 in the first study and on days 10 to 14 in the second study. Phenytoin, 55 mg/kg ip, was given on days 11 and 12 and on days 11 to 13 in the respective studies. In the first drinking water study, BSO enhanced the incidence of phenytoin-induced fetal resorptions 3.8-fold and cleft palates 3.3-fold (P less than 0.05) but did not affect postpartum death. In the second study, BSO enhanced the incidence of resorptions, cleft palates, and postpartum death by 2-fold, 2.6-fold, and 1.7-fold, respectively (P less than 0.05). In both of the latter two studies, phenytoin-induced fetal weight loss was altered by BSO treatment (P less than 0.05). BSO alone had no embryopathic effects. These results suggest that GSH may be involved in the detoxification of a reactive intermediate of phenytoin and/or in fetal cytoprotection.