The effect of phenytoin (PHT) on Na(+)-K(+)-ATPase and Mg(2+)-ATPase activities and on [14C]-PHT binding in vitro to synaptosomal and mitochondrial subcellular fractions from rat cerebral cortex was studied after chronic PHT treatment. Synaptosomal and mitochondrial fractions were characterized with plasma membrane and mitochondrial enzymatic markers. Synaptosomal Na(+)-K(+)-ATPase was not affected in vitro by PHT 1-200 microM or by chronic treatment with 2-50 mg/kg/day of the unlabeled drug for 8 days. Mitochondrial Mg(2+)-ATPase was significantly stimulated by PHT after chronic treatment with 5 mg/kg/day for 8 days; reaching maximal effect (76%), at 10-25 mg/kg. PHT had no effect on mitochondrial Mg(2+)-ATPase when added in vitro. [14C]-PHT binding in vitro to the subcellular fractions was determined by dialysis to assess in vivo binding of the unlabeled PHT during chronic treatment. Indeed, [14C]-PHT bound to synaptosomes was significantly reduced by chronic PHT treatment from 218 +/- 10 to 119 +/- 11 pmol/mg protein after 1 week of treatment; a similar effect was obtained after 2-3 weeks with 10 mg/kg/day. Mitochondrial fraction bound 117 +/- 10 pmol/mg protein labeled PHT. Chronic treatment with unlabeled PHT also reduced the amount of [14C]-PHT bound to 19.9 +/- 2.2 pmol/mg protein. These results show slow reversible PHT in vivo binding to synaptosomes and mitochondrias from rat cerebral cortex, supporting the idea that the modulatory action of PHT on Na+ and Ca2+ permeabilities are mediated through these slow reversible binding proteins. The data also suggest a possible role of intrasynaptosomal mitochondria in [Ca2+]i buffering.