The metabolism of CP-122,721, a neurokinin-1 antagonist, has been examined in vitro using hepatic microsomes from human and animal species, and recombinant heterologously expressed P450 enzymes. Metabolism occurs primarily via O-demethylation and N-dealkylation reactions. In human liver microsomes, O-demethylation was shown to be catalyzed by CYP2D6 with a low K(M) value. N-dealkyation was shown to be catalyzed primarily by CYP3A4. When scaled to in vivo, in vitro intrinsic clearance data yielded a reasonable correlation across species. CP-122,721 was shown to be metabolized by parallel pathways to 5-trifluoromethoxysalicylic acid, which had been observed as a major circulating metabolite in humans after oral administration of CP-122,721. The involvement of CYP1A2, CYP3A4, and MAO-B was demonstrated in the pathways leading to 5-trifluoromethoxysalicylic acid. The O-desmethyl metabolite of CP-122,721 was shown to undergo a P450 catalyzed O-detrifluoromethylation reaction yielding a p-hydroquinone metabolite. The reaction was shown to be catalyzed by CYP3A4. Incubation under (18)O(2) yielded the hydroquinone containing O-18, consistent with this reaction occurring via an ispo substitution mechanism. Combined, these findings provide a comprehensive understanding of the metabolism of this new agent.