The specific binding to alpha-adrenoceptors in crude plasma membrane preparations of the rat brain was studied by means of 3H-clonidine (specific radioactivity 26.7 Ci/mmole). Equilibrium binding of 3H-clonidine could be described adequately according to a two-site model with a minor population of high affinity sites (KD1 = 0.4 nM) and a major population of low affinity sites (KD2 = 6.1 nM). The heterogeneity of 3H-clonidine binding was also indicated by a biphasic association rate in kinetic binding studies. In competition experiments with 3H-clonidine concentrations of 0.5 or 4.0 nM respectively, concentration-dependent displacement was observed with the non-radioactive compounds: clonidine, B-HT 920 (2-amino-6-allyl-5, 6, 7, 8-tetrahydro-4H-thiazolo-[5, 4-d]-azepin-dehydrochloride) and B-HT 933 (2-amino-6-ethyl-4, 5, 7, 8-tetrahydro-6H-oxazolo-[5, 4-d]-azepin-dihydrochloride). IC50 values of 3, 21 and 160 nM or 10, 63 and 380 nM respectively were thereby evaluated. Cardiovascular effects were estimated in rats. The blood pressure increase in spinal animals was taken as parameter for alpha-adrenoceptor stimulation at peripheral vascular sites. The bradycardic effect in vagotomized animals was taken as parameter for central nervous sympathoinhibition. The ranking order of the potency of the three drugs was the same in both in vivo tests and parallels the in vitro binding affinities at both binding sites: clonidine greater than B-HT 920 greater than B-HT 933. These results indicate the similarity of the alpha-adrenoceptor structures in brain membrane preparations, at peripheral vascular sites and at central sympathoinhibitory sites.