Data are reported demonstrating a role for glutathione and age in the metabolic activation of phenytoin to a reactive metabolite. The in vitro liver microsomal covalent binding of [14C]-phenytoin (DPH) was examined in mice and rats. After incubation with 25-300 uM DPH, covalent binding was dose dependent and linear with time. Incubation in an atmosphere of carbon monoxide or nitrogen markedly decreased covalent binding. Comparison of covalent binding in male rats and mice pretreated with inducers of drug metabolism (phenobarbital, 3 -methylcholanthrene) showed significantly greater enhancement following phenobarbital induction compared to controls. In vitro addition of inhibitors of drug metabolism (piperonyl butoxide, α-naphthylisothiocyanate, cobaltous chloride, SKF-525A) all significantly decreased covalent binding. Binding studies with subcellular fractions showed maximal covalent binding in microsomes. Addition of thiols, i.e., glutathione, cysteine and cysteamine, significantly decreased covalent binding to 9-36% of control. Addition of butylated hydroxyanisole and butylated hydroxytoluene decreased covalent binding to 10-22% of control. In vivo pretreatment with diethyl maleate and in vitro preincubation with trichloropropene oxide resulted in a significant increase in covalent binding. Rats of ages 8 weeks, 24 weeks and 72 weeks showed a decrease both in covalent binding and in inducibility of covalent binding with increasing age. There was no significant difference in covalent binding between male and female rats of similar ages. These findings are consistent with a cytochrome P-450 dependent generation of a phenytoin arene oxide electrophilic arylating reactive intermediate.