The rostral ventrolateral medulla (RVLM) is the primary region maintaining vasomotor tone, and a site of action for central antihypertensive agents. In vitro [125I]p-iodoclonidine binding studies showed that moxonidine was selective for I1-imidazoline over α2-adrenergic receptors in the RVLM. We identified efaroxan and SK&F 86466 as selective I1- and α2-antagonists, respectively. We tested moxonidine's action within the RVLM of spontaneously hypertensive rats (SHRs) on I1-imidazoline or α2-adrenergic receptors, and determined whether the RVLM mediates the action of systemic moxonidine. SHRs were anesthetized, paralyzed, and ventilated and the RVLM was localized by testing for a pressor response to 2 nmol glutamate. To test whether I1 or α2 mediates hypotensive effects of moxonidine, the I1/α2 antagonist efaroxan (4 nmol) or the α2-blocker SK&F 86466 (10 nmol) was administered 15 min before 4 nmol moxonidine. Efaroxan elevated blood pressure and abolished the action of moxonidine, whereas α2-blockade with SK&F 86466 slightly lowered blood pressure and only partially attenuated moxonidine's effect. The depressor effect of intravenous moxonidine (40 μg/kg) was reversed within 10 min by microinjection of 10 nmol efaroxan into the RVLM. Prior bilateral microinjections of efaroxan (10 nmol in 80 nl/site) into the RVLM prevented the hypotensive action of moxonidine given i.v. (40 μg/kg). Pharmacokinetic studies showed that at the peak vasodepressor response (8 min post-injection), [3H]moxonidine spread less than 1 mm from the injection site. Moxonidine is a centrally acting antihypertensive with a selective action on I1-imidazoline receptors in RVLM.