Propafenone is a new class 1 antiarrhythmic agent. The drug is extensively metabolized. 5‐Hydroxylation andN‐dealkylation constitute major metabolic pathways. Recently it has been demonstrated that the in vivo metabolism of propafenone is controlled by the debrisoquin/sparteine polymorphism. To elucidate which of the above metabolic reactions is catalyzed by cytochrome P‐450db1, the formation of 5‐hydroxypropafenone andN‐desalkylpropafenone was studied in the microsomal fraction of four human kidney donor livers previously characterized with regard to their ability to hydroxylate the β‐adrenergic antagonist bufuralol. The l'hydroxylation of bufuralol is catalyzed by the P‐450db1responsible for polymorphic debrisoquin/sparteine oxidation. The formation of 5‐hydroxypropafenone but notN‐desalkylpropafenone was closely related to bufuralol l'hydroxylation. Incubation with LKM1 antibodies, which selectively recognize P‐450db1, inhibited 5‐hydroxypropafenone formation completely whereas N‐dealkylation was unimpaired. Propafenone was a strong competitive inhibitor of bufuralol l'hydroxylation. Thus it can be concluded that 5‐hydroxypropafenone is formed by the cytochrome P‐450 isozyme involved in polymorphic bufuralol oxidation.Clinical Pharmacology and Therapeutics(1989)45,2