Dehydrogenases that use ubiquinone as an electron acceptor, including complex I of the respiratory chain, complex II, and glycerol-3-phosphate dehydrogenase, are known to be direct generators of superoxide and/or H 2 O 2 . Dihydroorotate dehydrogenase oxidizes dihydroorotate to orotate and reduces ubiquinone to ubiquinol during pyrimidine metabolism, but it is unclear whether it produces superoxide and/or H 2 O 2 directly or does so only indirectly from other sites in the electron transport chain. Using mitochondria isolated from rat skeletal muscle we establish that dihydroorotate oxidation leads to superoxide/H 2 O 2 production at a fairly high rate of about 300pmol H 2 O 2 ·min −1 ·mg protein −1 when oxidation of ubiquinol is prevented and complex II is uninhibited. This H 2 O 2 production is abolished by brequinar or leflunomide, known inhibitors of dihydroorotate dehydrogenase. Eighty percent of this rate is indirect, originating from site II F of complex II, because it can be prevented by malonate or atpenin A5, inhibitors of complex II. In the presence of inhibitors of all known sites of superoxide/H 2 O 2 production (rotenone to inhibit sites in complex I (site I Q and, indirectly, site I F ), myxothiazol to inhibit site III Qo in complex III, and malonate plus atpenin A5 to inhibit site II F in complex II), dihydroorotate dehydrogenase generates superoxide/H 2 O 2 , at a small but significant rate (23pmol H 2 O 2 ·min −1 ·mg protein −1 ), from the ubiquinone-binding site. We conclude that dihydroorotate dehydrogenase can generate superoxide and/or H 2 O 2 directly at low rates and is also capable of indirect production at higher rates from other sites through its ability to reduce the ubiquinone pool.