1. The biotransformation of 3H-alfentanil was studied using suspension cultures of isolated hepatocytes of male and female rats and of dogs. 2. In hepatocytes of the male rat, alfentanil was readily metabolized, following linear Michaelis-Menten kinetics over the concentration range 5-400 microM. The metabolism was strongly inhibited by the cytochrome P-450 inhibitors metyrapone, alpha-naphthoflavone and piperonyl butoxide. 3. The major metabolites of alfentanil, which were formed in suspension cultures of male rat hepatocytes, were identified by h.p.l.c. co-chromatography and by mass spectrometry and included N-[4-(hydroxymethyl)-4-piperidinyl]-N-phenylpropanamide, N-[4-(methoxymethyl)-4-piperidinyl]-N-phenylpropanamide or noralfentanil and N-[1-[2-(4-ethyl-4,5-dihydro-5-oxo-1-H-tetrazol-1-yl)ethyl]- 4-(hydroxymethyl)-4-piperidinyl]-N-phenylpropanamide or desmethylalfentanil. 4. The major in-vitro metabolic pathways of alfentanil in hepatocytes of the three sources were oxidative N-dealkylation at the piperidine nitrogen and oxidative O-demethylation at the methoxymethyl moiety.