Objective: Endothelium-derived contracting factors (EDCFs) contribute to vascular dysfunction during hypertension. Here we test the hypothesis that AMPK-activation attenuates endoperoxide, EDCF-mediated vasoconstriction in aorta from hypertensive rats. Methods: Aortic rings from 6 SHR and 6 normotensive Wistar-Kyoto Rats (WKY) (20-24 weeks old) were isolated for vascular myography. Quiescent rings (not pre- constricted, set to resting tension) were exposed to increasing doses of acetylcholine (ACh) in the presence of NOS inhibition (L-NAME, 10^-4 M) and either no drug (vehicle only, CON), AICAR (2mM) and/or AMPK inhibitor Compound C (CC; 20mM). Endothelium- and endoperoxide-dependency was confirmed by removal of the endothelium (mechanically) or pre- treatment of rings with cyclooxygenase inhibitor indomethacin (10^-5 M). Western blots were performed on aortic rings snap frozen at the end of dose-response curve protocols to assess protein content and phosphorylation status across treatment groups. Results: In SHR, endothelium-dependent endoperoxide-mediated vasoconstriction to ACh was completely abolished in aortic rings pre-treated with AICAR (Area under the curve (AUC, arbitrary units) CON: 53±11, AICAR: 7±3, P < 0.01), indothemacin (AUC: 6±3, P < 0.01 vs. CON) or removal of the endothelium (AUC: 9±9, P < 0.01 vs. CON). Co-treatment of rings with AICAR + CC restored constriction to ACh (AUC: AICAR + CC: 41±7, P = NS vs. CON). Endothelium-dependent contractions were much weaker in WKY (AUC CON: 10±5, P < 0.01 vs SHR) and were not altered by any treatment. Western blots confirmed phospho-(Thr172)-AMPK and AMPK target phospho-(Ser79)-acetyl-CoA carboxylase were elevated in AICAR-treated SHR rings but not with AICAR + CC. Conclusions: These results suggest for the first time that AMPK stimulation inhibits over-active EDCF- mediated constriction in SHR aorta and thus establishes that AMPK may be a useful target to improve vascular function in hypertension.