Considerable disagreement still exists concerning the superoxide generation sites in the purified bovine heart NADH-ubiquinone oxidoreductase (complex I). Majority of investigators agree that superoxide is generated at the flavin site. Here we present a new hypothesis that the generation of superoxide reflects a dynamic balance between the flavosemiquinone (semiflavin or SF) and the semiquinone (SQ), like a "tug-of-war" through electrons. All preparations of bovine heart complex I, which have been isolated at Yoshikawa's laboratory, have one protein-bound endogenous ubiquinone per complex I (Shinzawa-Itoh et al., Biochemistry, 49 (2010) 487-492). Using these preparations, we measured (i) EPR signals of the SF, the SQ and iron-sulfur cluster N2 simultaneously with cryogenic EPR and (ii) superoxide production with both the room temperature spin-trapping technique and the partially acetylated cytochrome c method. Our experimental evidence was (1) without added decylubiquinone (DBQ), no catalytic oxidation of NADH occurs. The NADH addition produced mostly SF and it generated superoxide as reported by Kussmaul and Hirst (PNAS, 103 (2006) 7607-7612). (2) During catalytic electron transfer from NADH to DBQ, the superoxide generation site was mostly shifted to the SQ. (3) A quinone-pocket binding inhibitor (rotenone or piericidin A) inhibits the catalytic formation of the SQ, and it enhances the formation of SF and increases the overall superoxide generation. This suggests that if electron transfer was inhibited under pathological conditions, superoxide generation from the SF would be increased.