Alpha2-adrenergic inhibition of adenylate cyclase in bovine ciliary processes and rabbit iris ciliary body (ICB) was studied. With bovine ciliary process membrane, it was found that cAMP production in the presence of 1 μM isoproterenol was increased with increasing NaCl concentrations from 0 to 200 mM. Clonidine, an α2-adrenergic agonist, produced a NaCl concentration- dependent inhibition of cAMP production in the presence of isoproterenol with a maximum inhibition at 200 mM NaCl (P<0.05). NaCl concentrations had no effect on basal adenylate cyclase activities and activity in the presence of clonidine alone. The α2-adrenergic agonists, lofexidine, clonidine and p-amino-clonidine were tested for their ability to inhibit isoproterenol-stimulated adenylate cyclase in bovine ciliary process membrane in the presence of 200 mM NaCl. Their dose-response curves showed that they had similar IC50's but the maximum inhibition differed among these agonists. Clonidine was found to be a partial agonists producing 55% of the inhibition obtained with lofexidine. The specificity of inhibition of isoproterenol-stimulated adenylate cyclase by α2-agonists and blockade by pertussis toxin was examined by adenine labelling in rabbit ICB. The results demonstrate that α2-adrenergic receptors exert specific inhibitory effects on adenylate cyclase activity in rabbit ICB, which are mediated by an inhibitory guanine nucleotide regulatory protein, Gi.