The role of various subfamilies of rat hepatic cytochrome P450 in the oxidation of ethosuximide was evaluated by comparing ethosuximide clearance in control rats and those pretreated with relatively selective P450 inducers and/or inhibitors. Clotrimazole pretreatment increased ethosuximide clearance threefold (p less than 0.005). Dexamethasone increased ethosuximide clearance twofold (p less than 0.001), and the dexamethasone effect was completely abolished by a single dose of triacetyloleandomycin. These results suggest a prominent role for cytochrome P450IIIA in ethosuximide metabolism in the rat. Isoniazid increased ethosuximide clearance twofold (p less than 0.001), and this effect was abolished by a single dose of diallylsulfide, suggesting that ethosuximide is also processed by cytochrome P450IIE1 in rats. Phenobarbital pretreatment increased ethosuximide clearance 2-2.7 fold (p less than 0.001); an effect that was only partially reversed by orphenadrine, an inhibitor of cytochrome P450IIB/IIC enzymes. This suggests a quantitatively less important role for the IIB/IIC subfamilies in processing ethosuximide, since phenobarbital is an inducer of P450 subfamilies IIB, IIC, IIE, and IIIA. Neither the cytochrome P450IA inducer, beta-naphthoflavone, nor the inhibitor, alpha-naphthoflavone altered ethosuximide clearance. Ajmaline, an inhibitor of cytochrome P450IID, had no effect on ethosuximide clearance. Together, these findings suggest that ethosuximide is principally oxidized by cytochrome P450IIIA, and that cytochrome P450IIE may play an important role. Cytochromes P450IIB/C play less prominent roles in ethosuximide oxidation, and neither cytochrome P450IA nor cytochrome P450IID is involved.