Clonidine and other selective alpha-2 adrenergic agonists have been found to lower intraocular pressure in the eyes of rabbits and primates, including humans. It has been suggested that the ocular hypotensive response to alpha-2 agonists may be mediated, in part, by prejunctional inhibition of norepinephrine secretion at intraocular synapses. In this study, we have investigated the effects of adrenergic agonists and antagonists on field-stimulated, Ca++-dependent release of3H-norepinephrine (3H-NE) from isolated, perfused rabbit iris-ciliary bodies and have utilized radioligand binding methods to identify prejunctional adrenoceptors in this tissue. Clonidine (10−910−5M) produced a dosage-dependent inhibition of stimulation-evoked H-NE secretion (EC50≜3×10−8M), but did not alter basal secretion. Other adrenergic agonists capable of activating alpha-2 adrenoceptors (e.g., epinephrine, norepinephrine and xylazine) also significantly depressed3H-NE secretion, whereas selective alpha-1 adrenergic or beta adrenergic agonists were without effect. Clonidine-mediated inhibition of3H-NE release was reversed by the selective alpha-2 antagonist yohimbine (10−7M), but was unaffected by prazosin or timolol. Yohimbine alone markedly enhanced3H-NE secretion, indicating tonic activation of prejunctional alpha-2 adrenoceptors by endogenous released norepinephrine. Forskolin or 8-bromo-cAMP, which alone enhanced norepinephrine secretion, failed to attenuate the inhibitory responses to alpha-2 agonists.3H-rauwolsclne binding measurements showed a small decrease in alpha-2 receptor sites in iris-ciliary body membranes following surgical sympathetic denervation. It is concluded that the rabbit irisciliary body contains functional, prejunctional alpha-2 adrenoceptors which may play an autoregulatory rolein vivoand contribute to the ocular effects of adrenergic drugs.