To pursue the hypothesis that differences in the regulatory region of CYP2E1 are partially responsible for the intersubject variation in in vivo CYP2E1 activity, restriction enzyme digestion and Southern blotting of 36 human DNA samples were performed. The fractionated DNA was hybridized with a genomic probe to the upstream region of CYP2E1 from positions -2710 to -580. After digestion with the restriction enzyme XbaI, most subjects (69%) were homozygous for the expected band representing the XbaI fragment from positions -2270 to -408, whereas 31% had an unexpected, slightly larger band. Analyses of Southern blots of the same DNA samples cut by other restriction enzymes were consistent with the larger band containing an estimated 100-bp insertion and localized the mutation to a region from positions -2270 to -1672. To determine the functional significance of this mutation, in vivo CYP2E1 metabolic ability was determined in the same subjects using the 6-hydroxylation of chlorzoxazone as a probe. The presence of the insertion mutation was associated with greater CYP2E1 metabolic ability, but only among individuals who either were obese or had recently consumed ethanol (p < 0.01, both). These data are consistent with a DNA insertion that is associated with altered CYP2E1 induction. The incidence of the mutation was 31% among 65 African Americans and 6.9% among 58 Caucasians (p < 0.01). Thus, this CYP2E1 regulatory polymorphism not only enhances CYP2E1 metabolic ability, but is sufficiently common to impact susceptibility to CYP2E1-related diseases in at least two ethnic groups.