Through the use of an Escherichia coil 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 N-14 nucleus as found for menasemiquinone stabilized at the Q(D) site of E. coil 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.