An antitumor antibiotic, ascofuranone, selectively inhibited succinate- and NADPH-dependent O_2 uptake activity of the mito-chondria from Hansenula anomala, suggesting its interaction with the ubiquinone-reduction site of succinate dehydrogenase and external NADPH-dehydrogenase reactions. The action of ascofur-anone on the mitochondria is clearly different from that of antimycin A, the Q_i site inhibitor.