[3H]-guanfacine (N-amidino-2-(2,6-dichloro 3[3H] phenyl) acetamide hydrochloride; 24.2 Ci/mmol) has been used as a radioligand in homogenates of rat cerebral cortex. Specific binding of [3H]-guanfacine was linear with respect to tissue concentration (2.5-15 mg/ml), saturable and not markedly affected in the pH range 6.5-8.0. Analysis of the saturation of [3H]-guanfacine binding using an iterative least squares fitting procedure gave best fits to a single site model. [3H]-guanfacine binding was of high affinity (K(d) 1.77 ± 0.24 nM; n = 8) to a population of non interacting sites (nH 0.99 ± 0.02; n = 8) with a density of 118.2 ± 8.4 fmol/mg protein (n = 8). Highest levels of binding were achieved in cerebral cortex followed by thalamus > hypothalamus > medulla/pons > spinal cord > striatum >cerebellum. Binding was stereoselective with regard to the (-)-isomer of noradrenaline and the order of potency for displacement of [3H]-guanfacine by agonists was naphazoline > clonidine > (-)-adrenaline > (-)-α-methylnoradrenaline > (-)-noradrenaline > (±)-α-methylnoradrenaline > (+)-noradrenaline > methoxamine > (+)-adrenaline > phenylephrine and by antagonists was phentolamine > dihydroergocryptine > piperoxane > yohimbine > prazosin > labetalol > indoramin suggested binding to α2-adrenoceptors. The monovalent cations Na+ and K+ and also guanosine 5'-triphosphate (GTP) produced concentration-dependent inhibition whereas the divalent cations Ca2+, Mg2+, and Mn2+ first enhanced, then inhibited [3H]-guanfacine binding. Na+ (150 mM) or GTP (100 μM) produced marked reductions and Mn2+ (5 mM) marked increases in the number of receptor sites labelled by [3H]-guanfacine. It is concluded that [3H]-guanfacine preferentially labels a high affinity state of the α2-adrenoceptor in homogenates of rat cerebral cortex.