Through the use of an Escherichia coli strain deficient in menaquinone biosynthesis, purified nitrate reductase A (NarGHI)-enriched inner membrane vesicles were titrated and monitored by electron paramagnetic resonance (EPR) spectroscopy, revealing the formation of protein-bound ubisemiquinone (USQ) species. Two-dimensional ESEEM (HYSCORE) experiments on these radicals revealed the same magnetic interaction with an (14)N nucleus as found for menasemiquinone stabilized at the Q(D) site of E. coli NarGHI and assigned to His66 N(delta), a distal heme axial ligand. Moreover, this signature was lost in the NarGHI(H66Y) mutant, which is known to be unable to react with quinols. These findings demonstrate that NarGHI-bound USQ can be formed and detected by EPR. They also provide the first direct experimental evidence for similar binding of natural menasemiquinones and ubisemiquinones within the same protein Q site of NarGHI.