Chlorzoxazone is being developed and proposed for use as a probe to measure in vivo cytochrome P4502E1 activity, but the phenotypic trait measures that are used vary. Although the doses proposed for phenotyping range from 250 mg to 750 mg, the effect of dose on chlorzoxazone hydroxylation has not previously been evaluated. The purpose of this study was to characterize the pharmacokinetics of chlorzoxazone in normal healthy volunteers (N = 6) after single randomized oral doses of 250 mg and 750 mg. An additional 10 volunteers underwent a detailed pharmacokinetic study using the 250-mg dose to further evaluate proposed phenotypic trait measures (N = 16). Timed blood and urine samples were obtained for 10 hours for chlorzoxazone and 6-hydroxychlorzoxazone determination by HPLC. Pharmacokinetic parameter estimates were estimated using noncompartmental methods. Evaluation of phenotypic trait measures show that 6-hydroxychlorzoxazone to chlorzoxazone plasma concentration ratios at 2 to 4 hours after drug administration demonstrated the highest correlations with metabolite formation clearance (r = 0.9; P < 0.001). Urine-based parameters (e.g., total recovery) were not significantly related to formation clearance (r = 0.5; P > 0.05). Dose dependency in chlorzoxazone metabolism was shown by a 30% increase (P < 0.05) in the dose-normalized area under the concentration-time curve (AUC) of chlorzoxazone and lower incremental dose-normalized urinary recovery of 6-hydroxychlorzoxazone at early timepoints after the 750-mg dose. In addition, the plasma ratio of 6-hydroxychlorzoxazone to chlorzoxazone at 4 hours was reduced by 48% in 5 of 6 subjects after the 750-mg dose (P > 0.05). These data suggest that 6-hydroxylation was saturated at the higher dose and illustrate the importance of dose selection in phenotyping. The results of this study indicate that a chlorzoxazone dose of 250 mg should be used and that a single plasma ratio obtained 2 to 4 hours after dosing is reflective of chlorzoxazone 6-hydroxylation and thus may serve as a cytochrome P4502E1 phenotypic trait measure.