The biotransformation of midazolam is mediated by a cytochrome P-450 isozyme (P-450 IIIA) whose activity is highly variable. The kinetics of the 1'- and 4-hydroxylation of midazolam, the major routes of midazolam oxidation, by human liver microsomes have been examined to characterize further the cytochrome isozyme(s) catalysing these reactions, and to screen for drugs that might interfere with them. In hepatic microsomal preparation from two kidney donors (extensive and poor metabolisers of debrisoquine) KM values for 1'-hydroxylation were 4.2 and 6.1 microM (extensive and poor metabolisers, respectively), and for the 4-hydroxylation they were 14.7 and 18.1 microM, respectively. The corresponding Vmax values were 25.8 and 29.8 and 17.0 and 18.1 nmol.mg P-1.h-1. Both reactions appeared to be catalysed by the same or by coregulated isozymes. Midazolam hydroxylations in vitro are inhibited by many drugs, including nifedipine and other dihydropyridine-type calcium channel blockers, ergot alkaloids, cyclosporine, erythromycin and phenothiazine-type neuroleptics. A clinical case report illustrates the consequence of such a drug-drug interference with hepatic biotransformation; midazolam-induced sleep in a patient lasted for 6 days (t1/2 = 25 h).