Imidazoline binding sites are labeled by [3H]clonidine (I1) or by [3H]idazoxan (I2). I2-sites are mitochondrial. The subcellular localization of I1-sites in brain is unknown. Crude membranes from bovine rostral ventrolateral medulla (RVLM) were further purified by discontinuous sucrose density gradient. Fractions were assayed for I1-site density (Bmax) with [125I]p-iodoclonidine. Nonspecific binding was defined by 10 microM BDF-6143, and alpha 2-adrenergic binding was defined by 10 microM epinephrine. The proportions of I1 and alpha 2 in mitochondrial fractions were similar (28 +/- 3 and 24 +/- 4%, respectively), and both I1 and alpha 2 showed the greatest enrichment within the membrane-enriched fraction (58 +/- 13 and 38 +/- 4%). The myelin fraction contained a higher proportion of alpha 2 than I1 (38 +/- 4 and 15 +/- 2%), consistent with expression of alpha 2, but not I1, by glia. The enrichment of I1 and alpha 2 in cellular membranes and alpha 2 in myelin was confirmed by further purification of these fractions over a second discontinuous gradient. Following irreversible inactivation of alpha 2, the remaining I1 sites in RVLM crude membranes were inhibited by Gpp(NH)p but not by ATP. We conclude that I1-imidazoline sites are non-mitochondrial membrane proteins sensitive to guanine nucleotide and may be functional receptors.