A previously observed correlation between the rate of 6-hydroxylation of chlorzoxazone (CZX), a potent skeletal muscle relaxant, and cytochrome P450 2E1 activity in vitro led to the postulation that this drug may be used as a non-invasive probe for P450 2E1 activity in vivo. In this study, comparative pharmacokinetics of CZX and 6-hydroxychlorzoxazone (OH-CZX) were conducted in rats pretreated with an inhibitor or inducer of P450 2E1. After administration of CZX (150 mumol/kg, i.v.) to rats, blood samples were taken at different time points and the plasma concentrations of CZX and OH-CZX were determined by HPLC. The concentrations for CZX and OH-CZX over time were simultaneously fitted to a model of first-order elimination of CZX and first-order formation and elimination of OH-CZX using the computer program PCNONLIN to give pharmacokinetic parameters. Diallyl sulfide, a P450 2E1 inhibitor, at an oral dose of 50 or 200 mg/kg 12 hr prior to the CZX dose markedly inhibited the hydroxylation of CZX. Pretreatment with ethanol (15% in the drinking water for six days), a condition known to induce P450 2E1, slightly enhanced the formation of OH-CZX. To observe possible involvement of enzymes other than P450 2E1 in CZX metabolism, dexamethasone and phenobarbital were also used. Pretreatment with dexamethasone (50 mg/kg, i.p. daily for four days) did not cause changes in CZX and OH-CZX pharmacokinetics. Pretreatment with phenobarbital (75 mg/kg, i.p. daily for three days) enhanced CZX metabolism slightly. Our results suggest that P450 2E1 plays a major role in CZX hydroxylation in rats, but other factors may also be involved in the metabolism in vivo.