The synthetic opioid alfentanil (ALF) undergoes extensive metabolism via two major pathways: piperidine nitrogen dealkylation to noralfentanil (NA) and amide nitrogen dealkylation to N-phenylpropionamide (AMX). It is unknown whether AMX results from amide N-dealkylation of ALF directly, or indirectly from NA, the major metabolite of ALF. The major objectives of this investigation were to determine the metabolic origin of AMX and to identify the cytochrome P450 isoforms in human liver microsomes catalyzing ALF metabolism. Metabolites were quantitated by GC/MS. Significant amide N-dealkylation of ALF but not of NA by human liver microsomes was observed, indicating that AMX is derived directly from ALF and that there are two primary routes of ALF metabolism. Three strategies were used to identify the P450 isoform(s) catalyzing each of the two metabolic pathways: effect of isoform-selective inhibitors on metabolite formation catalyzed by human liver microsomes, correlation of metabolite formation rate with microsomal P450 isoform protein content and catalytic activity in a population of human livers, and metabolism by cDNA-expressed P450 isoforms. The mechanism-based P4503A4 inhibitor, troleandomycin, significantly inhibited formation of both NA and AMX. Other P4503A4 inhibitors, including midazolam, erythromycin, and ketoconazole, also diminished ALF metabolism to both metabolites. Formation rates of both NA and AMX were significantly correlated with microsomal P4503A4 protein content and catalytic activity. Of six expressed human P450 isoforms (P450s 1A2, 2A6, 2B6, 2D6, 2E1, and 3A4), only P4503A4 exhibited significant catalytic activity toward ALF dealkylation to NA and AMX. These results indicate the predominant role of P4503A4 in both major pathways of ALF metabolism.