OBJECTIVE: Comparison of a one-sample with a multi-sample method (the metabolic fractional clearance) to estimate CYP2E1 activity in humans. METHODS: Healthy, male Caucasians ( n=19) were included. The multi-sample fractional clearance (Cl(fe)) of chlorzoxazone was compared with one-time-point clearance estimation (Cl(est)) at 3, 4, 5 and 6 h. Furthermore, the metabolite/drug ratios (MRs) estimated from one-time-point samples at 1, 2, 3, 4, 5 and 6 h were compared with Cl(fe). RESULTS: The concordance between Cl(est) and Cl(fe) was highest at 6 h. The minimal mean prediction error (MPE) of Cl(est) as a percentage of actual mean Cl(fe) was -4.2% at 6 h. Furthermore, regarding Cl(fe), there was a negligible difference ( P=0.56) of bias between Cl(est) at 3 h (MPE=-8.9%) and 6 h (MPE=-4.2%). The best concordance between MR and Cl(fe) was found at 3 h (r=0.74; P<0.001). CONCLUSION: All three single-dose-sample estimates, Cl(est) at 3 h or 6 h, and MR at 3 h, can serve as reliable markers of CYP2E1 activity. The one-sample clearance method is an accurate, renal function-independent measure of the intrinsic activity; it is simple to use and easily applicable to humans.