Pimobendan, 4, 5-dihydro-6-(2-(4-methoxyphenyl)-1H-benzimidazol-5-yl)-5-methyl-3( 2-H )-pyridazinone, is a new inotropic drug that augments Ca(2+) sensitivity and inhibits phosphodiesterase in cardiomyocytes. Pimobendan is well absorbed after oral administration and is metabolized in the liver to the O-demethyl metabolite, which is also active. This study was conducted to identify the cytochrome P-450 (CYP) isoform(s) responsible for the pimobendan O-demethylation in human liver microsomes. Pimobendan O-demethylase activity in human liver microsomes was significantly correlated with phenacetin O-deethylase activity. CYP1A2 antibody and specific inhibitors of CYP1A2 strongly inhibited the metabolism of pimobendan. CYP1A2 was the only one of 10 recombinant human CYP isoforms tested that catalyzed pimobendan O-demethylation at the substrate concentration of 1 microM. At a high substrate concentration (100 microM), recombinant CYP3A4 also catalyzed the reaction, and antibody to CYP3A4 partially inhibited the activity in human liver microsomes. The contribution of CYP1A2 to pimobendan O-demethylation in human liver microsomes varied in the range of 18 to 76%, whereas CYP3A4 accounted for less than 10%, as calculated using the relative activity factor method. We conclude that CYP1A2 is one of the major enzymes responsible for the O-demethylation of pimobendan and CYP3A may make a minor contribution at clinically relevant concentrations of the drug.