The metabolism of alfentanil was studied in three healthy subjects after a 1-h infusion of 2.5 mg alfentanil-3H. One of the subjects was a poor hydroxylator of debrisoquine. Pharmacokinetic parameters were similar in the three subjects and were in the same range as those reported for volunteers. The majority of the administered radioactivity was excreted in the urine (90% of the dose), but unchanged alfentanil represented only 0.16-0.47% of the dose. Alfentanil and metabolites were characterized by HPLC co-chromatography with reference compounds and/or by mass spectrometry and quantified by GLC and radio-HPLC. The main metabolic pathway was N-dealkylation at the piperidine nitrogen, with formation of noralfentanil (30% of the dose). Other Phase I pathways were aromatic hydroxylation, N-dealkylation of the piperidine ring from the phenylpropanamide nitrogen, O-demethylation, and amide hydrolysis followed by N-acetylation. Glucuronic acid conjugation of aromatic or aliphatic hydroxyl functions was the main Phase II pathway. The second major metabolite was the glucuronide of N-(4-hydroxyphenyl) propanamide (14% of the dose). The metabolite pattern in these subjects was qualitatively very similar to that described previously in rats and dogs. Differences in the mass balance of urinary metabolites between the three subjects were very small, and there was no qualitative or quantitative evidence for a deficiency in the metabolism of alfentanil in the subject who was a poor metabolizer of debrisoquine.