Biotransformation of the imidazobenzodiazepine midazolam to its alpha-hydroxy and 4-hydroxy metabolites was studied in vitro using human liver microsomal preparations. Formation of alpha-hydroxy-midazolam was a high-affinity (Km = 3.3 mumol/L) Michaelis-Menten process coupled with substrate inhibition at high concentrations of midazolam. Formation of 4-hydroxy-midazolam had much lower apparent affinity (57 mumol/L), with minimal evidence of substrate inhibition. Based on comparison of Vmax/Km ratios for the two pathways, alpha-hydroxy-midazolam formation was estimated to account for 95% of net intrinsic clearance. Three azole antifungal agents were inhibitors of midazolam metabolism in vitro, with inhibition being largely consistent with a competitive mechanism. Mean competitive inhibition constants (Ki) versus alpha-hydroxy-midazolam formation were 0.0037 mumol/L for ketoconazole, 0.27 mumol/L for itraconazole, and 1.27 mumol/L for fluconazole. An in vitro-in vivo scaling model predicted inhibition of oral midazolam clearance due to coadministration of ketoconazole or itraconazole; the predicted inhibition was consistent with observed interactions in clinical pharmacokinetic studies. The selective serotonin reuptake inhibitor (SSRI) antidepressant fluoxetine and its principal metabolite, norfluoxetine, also were inhibitors of both pathways of midazolam biotransformation, with norfluoxetine being a much more potent inhibitor than was fluoxetine itself. This finding is consistent with results of other in vitro studies and of clinical studies, indicating that fluoxetine, largely via its metabolite norfluoxetine, may impair clearance of P450-3A substrates.