The technique of distance measurement utilizing spin relaxation enhancement by an external probe has been extended to the study of intrinsic semiquinone radicals through the use of holmium-EDTA complexes and continuous wave electron paramagnetic resonance spectroscopy. This technique has been used to determine the distance of the semiquinone anion. Q 1 (also designate as Q n − or Q c −), from the surface of the ubiquinone cytochrome c oxidoreductase, consisting of only three subunits, in membrane particles from Rhodobacter capsulates. The location of the semiquinone anion is 6–10Åfrom the N side protein, establishing that there are two separate quinone reaction sites, i.e., ‘Q 1’ and ‘Q 0’, within this complex on opposite sides of the membrane. The results are discussed in relation to reported ENDOR, EPR, and optical studies of the mitochondrial counterpart.