The specific binding of [3H]clonidine was used to characterize alpha-2 adrenoreceptors on rat tail artery smooth muscle membranes. At 24 degrees C binding of 8 nM [3H]clonidine was rapid T 1/2 of association = 2.1 min) and reversible (T 1/2 of dissociation = 0.7 min). The binding sites for the [3H]clonidine showed the specificity required for the alpha-2 adrenoreceptor. The rank order of potency of inhibitors of [3H]clonidine binding for agonists was clonidine greater than phenylephrine greater than methoxamine and for antagonists was yohimbine and piperoxan much greater than prazosin. Scatchard analysis of the binding data indicated the existence of a single population of binding sites with the maximum number of binding sites, Bmax, equal to 33.5 +/- 0.3 fmoles/mg of protein and the dissociation constant, KD, equal to 7.3 +/- 0.4 nM. There was no evidence for cooperativity (Hill coefficient = 0.96). The inhibition constants, Ki values, of various adrenergic agonists and antagonists for the displacement of [3H]clonidine from tail artery membranes were well correlated with Ki values determined for the displacement of this ligand from rat hippocampal membranes. Similar correlations were found with the potencies of these same agents in producing contractions of isolated tail artery strips. This study confirms the presence of alpha-2 adrenoreceptors on arterial smooth muscle and suggests that these receptors might be important in vascular function.