The Na+-pumping NADH-ubiquinone oxidoreductase has six polypeptide subunits (NqrA-F) and a number of redox cofactors including: a non-covalently bound FAD and a 2Fe-2S center in subunit F, covalently bound FMN’s in subunits B and C, and a non-covalently bound riboflavin in an undisclosed location. The FMN cofactors in subunits B and C are bound to threonine residues by phosphoester linkages. A neutral flavin-semiquinone radical is observed in the oxidized enzyme, while an anionic flavin-semiquinone has been reported in the reduced enzyme. For this work, we have altered the binding ligands of the FMN’s in subunits B and C by replacing the threonine ligands with other amino acids, and studied the resulting mutants by EPR and ENDOR spectroscopy. We conclude that Na+-NQR forms three spectroscopically distinct flavin radicals: 1) a neutral radical in the oxidized enzyme, which is observed in all of the mutants, and most likely arises from the riboflavin, 2) an anionic radical observed in the fully-reduced enzyme, which is present in wild type, and the NqrC-T225Y mutant, but not the NqrB-T236Y mutant, 3) a second anionic radical, seen primarily under weakly reducing conditions, which is present in wild type, and the NqrB-T236Y mutant but not the NqrC-T225Y mutant. Thus, we can tentatively assign the first anionic radical to the FMN in subunit B and the second to the FMN in subunit C. The second anionic radical has not been previously reported. In ENDOR spectra, it exhibits a larger linewidth and larger 8a-methyl proton splittings, compared to the first anionic radical.