Cilostazol (OPC-13013; 6-[4-(1-cyclohexl-1H-tetrazol-5-yl)butoxy]-3,4- dihydro-2(1H)-quinolinone) is widely used as an antiplatelet vasodilator agent. In vitro, the hydroxylation of the quinone moiety of cilostazol to OPC-13326 [6-[4-(1-cyclohexyl-1H-tetrazol-5-yl)butoxy]-3,4-dihydro-4-hydroxy-2(1H) -quinolinone], is the predominant route, and the hydroxylation of the hexane moiety to OPC-13217 is the second most predominant route. This study was carried out to identify and kinetically characterize the human cytochrome P450 (P450) isozymes responsible for the formation of the two major metabolites of cilostazol, namely, OPC-13326 and OPC-13217 [3,4-dihydro-6-[4-[1-(cis-4- hydroxycyclohexyl)-1H-tetrazol-5-yl)butoxy]-2(1H)-quinolinone)]. In in vitro studies using 14 recombinant human P450 isozymes, CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP2J2, CYP3A4, CYP3A5, and CYP4A11, cilostazol was metabolized to OPC-13326 mainly by CYP3A4 (K m = 5.26 μM, intrinsic clearance (CLint) = 0.34 μl/pmol P450/min), CYP1B1 (Km = 11.2 μM, CLint = 0.03 μl/pmol P450/min), and CYP3A5 (Km = 2.89 μM, CL int = 0.05 μl/pmol P450/min) and to OPC-13217 mainly by CYP3A5 (Km = 1.60 μM, CLint = 0.57 μl/pmol P450/min), CYP2C19 (Km = 5.95 μM, CLint = 0.16 μl/pmol P450/min), CYP3A4 (Km = 5.35 μM, CLint = 0.10 μl/pmol P450/min), and CYP2C8 (Km = 33.8 μM, CLint = 0.009 μl/pmol P450/min). The present study showed that the two major metabolites of cilostazol in vitro, namely, OPC-13326 and OPC-13217, are mainly catalyzed by CYP3A4 and CYP3A5, respectively.