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Ubiquinol-15 can be used as an electron donor for both the conventional cytochrome chain and the chloramphenicol-induced alternative pathway of Neurospora crassa mitochondria. The oxidation of ubiquinol-15 through the chloramphenicol-induced cyanide-insensitive pathway is not inhibited by antimycin A and is strongly inhibited by salicylhydroxamic acid. In contrast, the oxidation of duroquinol by N. crassa mitochondria is sensitive to antimycin A under all circumstances, except for some portion which is believed to be a non-enzymic autoxidation reaction. 2-Thenoyltrifluoroacetone, a specific inhibitor of the succinate-ubiquinone oxidoreductase activity of the respiratory chain, was found to be a potent inhibitor of the chloramphenicol-induced alternative oxidase-mediated NADH and ubiquinol-15 oxidation in N. crassa mitochondria. It may be concluded that 2-thenoyltrifluoroacetone interacts with a specific electron carrier of the alternative oxidase, possibly an iron-sulphur centre associated with an ubisemiquinone.