The ability of human cytochromes P450 other than CYP2E1 to catalyse the 6-hydroxylation of chlorzoxazone (6-OH-CHZ) was examined in vitro using human liver microsomal preparations and in vivo using chlorzoxazone as a metabolic probe. Chlorzoxazone 6-hydroxylation activity was significantly correlated with 4-nitrophenol 2-hydroxylase activity and immunodetected CYP2E1 in 14 human liver samples (r = 0.92 and 0.81, P < 0.001, respectively). Conversely, this catalytic activity was not correlated with CYP 3A or CYP1A activities. Diethyldithiocarbamate (DEDTC), a specific CYP2E1 inhibitor, reduced chlorzoxazone 6-hydroxylase activity by 92.3 ± 7.6% (n = 14 samples) while ketoconazole, a specific CYP3A inhibitor, reduced this activity by 8.6 ± 6.3% (n = 14). The residual activity following preincubation with DEDTC was significantly correlated with nifedipine oxidation and tamoxifen N-demethylations, both specific to CYP3A (r = 0.76 and 0.68, respectively). Genetically produced pure human CYP2E1 and 3A4 hydroxylated chlorzoxazone with turnover numbers of 19.7 and 0.14 min-1, respectively. Furthermore, cytochrome b5 stimulated chlorzoxazone 6-hydroxylation. From examination of the relative liver content of CYP2E1 and 3A, it can be asserted that CYP2E1 is the major enzyme involved in chlorzoxazone 6-hydroxylation and that the contribution of CYP3A is very minor. CYP2E1 activity was evaluated by the plasmatic metabolic ratio 6-OH-CHZ/CHZ (CHZ-MR) measured 2 h after ingestion of 500 mg CHZ. Smoker status did not influence the rate of CHZ hydroxylation. The CHZ-MR was 0.30 ± 0.13 (mean ± SD) n = 39 non-smokers versus 0.32 ± 0.15, n = 75 smokers. This result suggests that CYP1A, inducible by cigarette smoking, is not significantly involved in chlorzoxazone hydroxylation. Women exhibited a slightly lower CHZ-MR than men (0.29 ± 0.15, n = 44 versus 0.34 ± 0.15 n = 49, respectively). Obesity increased CHZ-MR, especially in non-insulindependent diabetic individuals (0.45 ± 0.21, n = 13 versus 0.30 ± 0.15, n = 42 control individuals, P = 0.007). Furthermore, exposure of workers to volatile organics in a shoe factory decreased CHZ-MR (0.19 ± 0.09, n = 10 Mexican workers versus 0.34 ± 0.12, n = 16 Mexican control individuals, P = 0.001). Concomitant administration of grapefruit juice (known to be an inhibitor of CYP3A4) with chlorzoxazone did not significantly modify the CHZ metabolic ratio: 0.29 ± 0.1 versus 0.31 ± 0.1, for nine control individuals without and with grapefruit juice, respectively. In conclusion, all these results demonstrate that chlorzoxazone is a very selective probe for phenotyping CYP2E1 in humans.