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NADH-quinone oxidoreductase is classified into two groups, NADH dehydrogenase-1 (NDH-1) and NADH dehydrogenase-2 (NDH-2). Animal mitochondrial complex I is an NDH-1 type enzyme. Previously, we isolated potent inhibitors from plants to both NDH-1 and NDH-2. We have now examined detailed inhibitory effects of three tannins (pentagalloylglucose, sanguiin H-11,and oolonghomobisflavan A) on NDH-1 using bovine heart mitochondrial complex I and a subcomplex flavoprotein (containing 3 subunits) derived from complex I. Although many specific inhibitors of NDH-1 (e.g. rotenone and piericidin A) have been reported, the reactive sites are at or near to, the ubiquinone-binding site. NADH-ubiquinone-1 oxidoreductase activity of complex I was inhibited by the three tannins, among which sanguiin H-11 was the most potent inhibitor. NADH-menadione oxidoreductase activity of complex I was susceptible to the three tannins, but completely resistant to rotenone. The inhibitory effects of tannins were all noncompetitive with respect to NADH, ubiquinone-1,and menadione. The NADH-menadione oxidoreductase of flavoprotein was also inhibited by the three tannins, but not by rotenone, which is consistent with the fact that flavoprotein does not contain a native ubiquinone-binding site. The study of the NADH reduced-minus-oxidized difference spectrum of flavoprotein under steady-state conditions indicated that the inhibitory sites of sangiin H-11 and oolonghomobisflavan A exist between the NADH binding site and the FMN site, and that for pentagalloylglucose exists between FMN and an artificial electron acceptor-binding site. These results suggest that the tannins are potent inhibitors of NADH dehydrogenases, and that the inhibitory mechanisms are novel.