In the present study, we have employed the monoradioiodinated alpha 2-agonist clonidine ([125I]-CLO) to characterize duck hypothalamic alpha 2-adrenoceptors and to localize alpha 2-specific binding sites in the duck brain. To validate the alpha 2-specificity of [125I]-CLO using an enriched duck hypothalamic membrane fraction, a radioreceptor assay was established by altering the membrane protein concentration, time, temperature and ionic milieu of incubation, and in the presence or absence of protease inhibitors. Competitive displacement studies revealed the following sequence of potency to displace [125I]-CLO: yohimbine greater than (-)-epinephrine greater than clonidine greater than (-)-norepinephrine greater than phentolamine greater than (-)-phenylephrine greater than (-)-isoproterenol greater than prazosin. The non-hydrolyzable guanosine 5'-triphosphate analog guanylylimidodiphosphate markedly inhibited [125I]-CLO binding suggestive of G-protein involvement. With regard to the histological distribution, diencephalic structures, such as the habenula and the nucleus reticularis of the thalamus, were densely labeled by [125I]-CLO. In the hypothalamus, alpha 2-adrenoceptors were detected in the antidiuretic hormone-synthesizing nucleus paraventricularis, the nucleus praeopticus medialis, the nucleus anterior medialis hypothalami, the nucleus magnocellularis praeopticus, the nucleus commissurae pallii, the nucleus inferior hypothalami and the regio lateralis hypothalami. Circumventricular organs, such as the plexus choroidei, organum subfornicale, organum paraventriculare and the corpus pineale, were endowed with alpha 2-specific binding sites, as were the cell layers of the tectum opticum. In addition, telencephalic structures revealed high receptor densities.(ABSTRACT TRUNCATED AT 250 WORDS)