The alpha-2 adrenoceptor subtype and its signal transduction pathway mediating vascular relaxation in rats were studied in vitro using rings of superior mesenteric arteries. Removal of endothelium or incubation with NG-nitro-L-arginine completely blocked relaxant responses to UK14,304, suggesting endothelium-derived nitric oxide mediates relaxation. The order of potency for full (F) or partial (P) agonists causing relaxation was guanabenz (P) > UK14,304 (F) > clonidine (P) > epinephrine (F) > norepinephrine (F). Affinities (Ka) of alpha-2 adrenoceptor subtype-selective drugs for blocking relaxation were obtained in side-by-side experiments comparing rat mesenteric arteries with pig coronary arteries. Relaxation of pig coronary arteries is known to be mediated by the alpha-2A adrenoceptor subtype. Ka values in nM for rauwolscine (19), WB-4101 (265), SKF-104078 (197), spiroxatrine (128), and prazosin (1531) for blocking relaxation in rat arteries were consistent with their affinities for binding at the alpha-2D adrenoceptor subtype. Ka values for rauwolscine and WB-4101, drugs distinguishing the alpha-2D from the alpha-2A adrenoceptor subtype, were significantly higher in blocking relaxation of rat arteries compared with pig arteries, suggesting the alpha-2D adrenoceptor subtype mediates NO-induced relaxation in rat arteries. We used forskolin to oppose alpha-2 adrenoceptor-mediated inhibition of cAMP formation by directly stimulating cAMP formation in endothelium. Forskolin did not affect the relaxant response to UK14,304, suggesting that cAMP is not involved in the coupling of alpha-2 adrenoceptors to nitric oxide-induced vascular relaxation.