1. The relative roles of human hepatic cytochrome P450 (CYP) subfamilies participating in ethosuximide metabolism have been studied in vitro using humanized heterologous CYP microsomal systems expressing either CYP1A2, CYP2A6, CYP2C9, CYP2D6, CYP2E1 or CYP3A4. 2. Ethosuximide was incubated with each expression system at 37 degrees C, and its hydroxylated metabolite was quantified by HPLC assay. 3. K(m) and V(max) values for metabolite formation were estimated for CYP3A4 and CYP2E1. The K(m) values for the metabolite formed were 1.40 and 0.24 mM for CYP3A4 and CYP2E1, respectively. The V(max) values were 0.65 and 0.14 nmol mg-1 protein min(-1) for CYP3A4 and CYP2E1, respectively. 4. These parameters could not be measured for other enzymes, since metabolite concentrations formed were below the HPLC detection limits. 5. Immuno-inhibition studies using specific antibodies against CYP3A4 or CYP2E1 revealed that ethosuximide metabolite levels decreased when the amount of added CYP3A4 or CYP2E1 antibody increased, with anti-CYP3A4 antibodies yielding a greater inhibitory effect. 6. Simulations of scaled-up in vivo ethosuximide CL(hepatic) mediated by CYP3A4 and CYP2E1 based on the in vitro CL'(int) values, which were calculated from the foregoing respective V(max) and K(m) values, project that well over 90% of in vivo CL(hepatic) is due to CYP3A4. These results support an important role for CYP3A in human ethosuximide metabolism, and a minor role for CYP2E1.