The biotransformation of sufentanil (SUF), an analog of the synthetic opioids fentanyl and alfentanil, was investigated in liver microsomes of rats, dogs, and humans. The drug was extensively metabolized and the metabolism was found to be very similar, both kinetically and metabolically, in the three species. The initial metabolism of SUF occurred monophasically in man and dog and biphasically in the rat over a concentration range of 0.13-20.1 microM. The apparent Vm values were 7.30 and 6.15 nmol metabolized.min-1.mg protein-1, and the apparent Km values were 4.98 microM and 15.2 microM for dog and human microsomes, respectively. In rat microsomes, apparent Km values were 0.10 and 20.8 microM, and the apparent Vm values were 0.10 and 7.32 nmol metabolized.min-1.mg protein-1 for the high and low affinity site, respectively. The major metabolic pathways were similar in the three species and included oxidative N-dealkylation at the piperidine nitrogen, oxidative N-dealkylation of the piperidine ring from the phenylpropanamide nitrogen, oxidative O-demethylation, and aromatic hydroxylation. Desmethyl-SUF was formed at the shorter incubation times but quickly metabolized into secondary metabolites. The major metabolites which could be detected at the end of the incubation were N-[4-(methoxymethyl)-4-piperidinyl]-N-phenylpropanamide, N-[4-(hydroxymethyl)-4-piperidinyl]-N-phenylpropanamide, and N-phenylpropanamide. The relevance of the in vitro results is discussed in relation to previous in vivo studies of the metabolism of SUF in rats, dogs, and humans.