Summary: Both the hypotension and the sedation elicited by centrally acting antihypertensive agents traditionally are ascribed to activation of α2-adrenergic receptors. However, second-generation centrally acting antihypertensive agents such as moxonidine and rilmenidine are less sedating but retain antihypertensive efficacy. The vasodepressor action of clonidine within its brainstem site of action in the ventrolateral medulla has been linked to a novel receptor that recognizes imidazoles and related compounds. We sought to characterize the interactions of moxonidine with these putative I1-imidazoline receptors. Moxonidine showed 40- to 70-fold selectivity for I1-imidazoline over α2-sites in the ventrolateral medulla, as shown by nonlinear curve-fitting and by masking studies using epinephrine and imidazole-4-acetic acid to block α2-receptors and I1-imidazoline sites, respectively. In the rat renal medulla, moxonidine showed greater than 600-fold selectivity for renal imidazoline sites relative to the α2B-adrenergic subtype. The high affinity of moxonidine for I1-imidazoline binding sites was confirmed by using membranes prepared from bovine adrenomedullary chromaffin cells, which lack α2-adrenergic receptors, In comparison with rilmenidine, another second-generation agent, moxonidine, showed higher affinity at I1-imidazoline receptors and higher selectivity relative to α2-adrenergic receptors. Binding studies using [3H]moxonidine support the conclusion that moxonidine is a selective high-affinity ligand for the putative I1-imidazoline receptor. Moxonidine exhibits a unique combination of high affinity and selectivity toward the I1-imidazoline receptor and may be a useful pharmacological probe for further studies of imidazole receptor function as well as a promising new therapeutic agent.