This study has determined the subtype(s) of postsynaptic alpha 2-adrenoceptors in rat brain. This question has been addressed by using two separate approaches, i.e. ligand displacement of [3H]2-(2-methoxy)-1,4-benzodioxan-2-yl)-2-imidazoline ([3H]RX 821002) from membranes prepared from rat cortex after noradrenergic denervation and, secondly, by antagonism of clonidine-induced mydriasis. After rats had been lesioned using N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4; 100 mg/kg i.p., 30 min after zimeldine 10 mg/kg i.p.), noradrenaline was undetectable in the cortex 3 days later. Displacement of [3H]RX 821002 with a range of agonists and antagonists which distinguish between the known alpha 2-adrenoceptor subtypes (alpha 2A-2D) yielded pKi values which correlated very well with reported values for the alpha 2D-adrenoceptor (r = 0.929; P < 0.001), but not the alpha 2A (r = 0.450; P = 0.192), alpha 2B (r = 0.280, P = 0.434) or alpha 2C (r = 0.283; P = 0.460) subtypes. Similarly, the potencies of various alpha 2-adrenoceptor antagonists to inhibit clonidine (0.03 mg/kg i.p.)-induced mydriasis in conscious rats correlated strongly with their pKi values for alpha 2D-adrenoceptors (r = 0.899; P = 0.015) but not alpha 2A-(r = 0.369; P = 0.472), alpha 2B-(r = -0.224; P = 0.670) or alpha 2C-adrenoceptors (r = 0.253; P = 0.584). These data are, therefore, consistent and argue strongly that postsynaptic alpha 2-adrenoceptors in the rat cortex and Edinger-Westphal nucleus are of the alpha 2D subtype.