Biotransformation of the triazolobenzodiazepine alprazolam (ALP) was studied in vitro using hepatic microsomal preparations from human, monkey, mouse, and rat liver tissue. Two principal hydroxylated metabolites were identified: 4-hydroxy- and α-hydroxy-alprazolam (4-OH-ALP and α-OH-ALP). In all species, rates of 4-OH-ALP formation exceeded those of α-OH-ALP. In human liver microsomes, ratios of 4-OH-ALP/α-OH-ALP reaction velocities calculated at clinically relevant plasma concentrations of ALP ranged from 7 to 17, qualitatively consistent with, but numerically larger than, the ratio of the plasma levels of the two metabolites during clinical use of ALP in humans. Km values for both 4-OH-ALP (170–305 µM) and α-OH-ALP (63–441 µM) considerably exceeded the usual maximum plasma concentration observed in humans (200 ng/ml, 0.65 µM), consistent with the linear (dose-independent) pharmacokinetic characteristics of ALP observed in humans. Thus formation of 4-OH-ALP via hydroxylation is the major route of ALP metabolism. This pathway is probably mediated by the cytochrome P-450-3A subfamily. Factors that impair the activity of this cytochrome subtype are likely to impair clearance of ALP in vivo.