The cytochrome P450 3A (CYP3A) subfamily (mainly CYP3A4 and CYP3A5) is responsible for metabolizing approximately half of currently marketed drugs, but with considerable interindividual variability in expression and function. To investigate factors contributing to this variability, rates of midazolam (MDZ) 1'-hydroxylation and CYP3A4 and CYP3A5 protein content were determined using a set of 54 human liver microsomes (HLMs). Genetic factors investigated included CYP3A4 and CYP3A5 single nucleotide polymorphisms (SNPs) and haplotypes, CYP3A4 mRNA alternative splicing, and CYP3A4 transcriptional start and polyadenylation sites. Demographic and environmental factors investigated included age, gender, and histories of smoking and alcohol consumption. MDZ 1'-hydroxylation rates varied from 0.025 to 3.106 nmol/min/mg protein, with significant correlation to CYP3A4 protein content (r(s) = 0.92, P < 0.001) but less robust correlation to CYP3A5 protein content (r(s) = 0.60, P < 0.001). We identified eight CYP3A4 SNPs (five novel) and nine CYP3A5 SNPs (one novel), as well as seven CYP3A4 and two CYP3A5 haplotypes (all novel). No influence of genotype or haplotype on MDZ 1'-hydroxylation rate was observed, although CYP3A5*3A (g.6986a>g; g.31611c>t) carriers had lower CYP3A5 protein content compared with noncarriers (P = 0.004). No alternative splicing of CYP3A4 mRNA was found. Likewise, only a single transcriptional start site and polyadenylation site for CYP3A4 mRNA were identified. Subjects with a history of alcohol consumption had 2.2-fold higher median MDZ 1'-hydroxylation (P = 0.017), whereas no influence of age, gender, or smoking was evident. In conclusion, the investigated genetic factors did not contribute substantially to the large interindividual variability in midazolam hydroxylation, although alcohol consumption has a discernable but modest influence.