The 6-hydroxylation of chlorzoxazone (CLZ) is currently being used in both in vivo and in vitro studies to quantify cytochrome P450 2E1 (CYP2E1) activity in humans. Comparatively little is known with regard to the biotransformation of this drug in other species. The NADPH-dependent biotransformation of CLZ was therefore studied using hepatic microsomes derived from humans and ten other mammalian species. In all species, 6-hydroxychlorzoxazone (6OH-CLZ) was the only metabolic product that could be identified by HPLC with ultraviolet detection. Enzyme kinetic analysis was used to characterize this CLZ 6-hydroxylase activity. Although the majority of kinetic data conformed to a single-enzyme Michaelis-Menten model, a two-enzyme (high and low affinity) model was required for four species (ferret, monkey, pig, and rat). Apparent K(m) values for the high-affinity component ranged from 12 microM (pig) to 95 microM (rabbit). The rank order of Vmax/K(m), an index of intrinsic clearance, was: mouse > horse > monkey > rabbit > cow > ferret > pig > human 1 > rat > human 2 > cat > dog. Diethyldithiocarbamate (DDC), a CYP2E1 inhibitor in humans, was a potent mechanism-based inhibitor of 6OH-CLZ formation in microsomes from all species examined. Preincubation of microsomes for 15 min in the presence of DDC and NADPH significantly enhanced the maximum degree of inhibition but had no effect on inhibitor potency. Inhibitor concentrations at 50% of maximum inhibition (IC50max) for DDC with preincubation ranged from 9 microM (human) to 45 microM (cow). In conclusion, DDC-sensitive CLZ 6-hydroxylation was identified as the principal NADPH-dependent pathway for chlorzoxazone metabolism in liver microsomes from humans and ten other mammalian species. These data indicate a species-conserved mechanism for the oxidative biotransformation of chlorzoxazone.