The enzyme kinetics of the initial hydroxylation of ethylbenzene to form 1-phenylethanol were determined in human liver microsomes. The individual cytochrome P450 (CYP) forms catalysing this reaction were identified using selective inhibitors and recombinant preparations of hepatic CYPs. Production of 1-phenylethanol in hepatic microsomes exhibited biphasic kinetics with a high affinity, low K m, component (mean K m=8 μM; V max=689 pmol/min/mg protein; n=6 livers) and a low affinity, high K m, component ( K m=391 μM; V max=3039 pmol/min/mg protein; n=6). The high-affinity component was inhibited 79%–95% (mean 86%) by diethyldithiocarbamate, and recombinant CYP2E1 was shown to metabolise ethylbenzene with low K m (35 μM), but also low V max (7 pmol/min/pmol P450), indicating that this isoform catalysed the high-affinity component. Recombinant CYP1A2 and CYP2B6 exhibited high V max (88 and 71 pmol/min/pmol P450, respectively) and high K m (502 and 219 μM, respectively), suggesting their involvement in catalysing the low-affinity component. This study has demonstrated that CYP2E1 is the major enzyme responsible for high-affinity side chain hydroxylation of ethylbenzene in human liver microsomes. Activity of this enzyme in the population is highly variable due to induction or inhibition by physiological factors, chemicals in the diet or some pharmaceuticals. This variability can be incorporated into the risk assessment process to improve the setting of occupational exposure limits and guidance values for biological monitoring.