The imidazoline-guanidinium receptive site (IGRS) is a membrane-bound protein that may mediate some of the pharmacological effects of imidazoline and guanidinium compounds. The structure and functionality of this protein are unknown but, in addition to its location at the plasma membrane, it is found in high density in the outer membrane of mitochondria (Tesson, F., Prip-Buus, C., Lemoine, A., Pegorier, J.-P., and Parini, A. (1991) J. Biol. Chem. 266, 155-160). Using a two-step procedure, we report the purification of mitochondrial IGRS from rabbit kidney to the apparent homogeneity. After solubilization of mitochondrial membranes with digitonin, an apparently homogeneous IGRS preparation was obtained by two sequential purification steps, chromatofocusing and hydroxylapatite-agarose chromatography. One- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the purified preparation after silver staining or radioiodination indicated that IGRS binding subunit was purified at the apparent homogeneity since a single band (M(r) approximately 60,000) was observed. IGRS behaves as an acidic protein (pI 5.5) whose binding activity is regulated by H+ concentration near a physiological pH of 7.4. The ability to achieve rapid purification of IGRS should facilitate efforts to define molecular properties and functionality of this protein.