K_ATP channels are involved in regulating coronary function, but the contribution of endothelial K_ATP channels remains largely uncharacterized. We generated a transgenic mouse model to specifically target endothelial K_ATP channels by expressing a dominant negative Kir6.1 subunit only in the endothelium. These animals had no obvious overt phenotype and no early mortality. Histologically, the coronary endothe‐lium in these animals was preserved. There was no evidence of increased susceptibility to ergonovine‐in‐duced coronary vasospasm. However, isolated hearts from these animals had a substantially elevated basal coronary perfusion pressure. The K_ATP channel openers, adenosine and levcromakalim, decreased the perfusion pressure whereas the K_ATP channel blocker glibenclamide failed to produce a vasoconstrictive response. The inducible endothelial nitric oxide pathway was intact, as evidenced by vasodilation caused by bradykinin. In contrast, basal endothelin‐1 release was significantly elevated in the coronary effluent from these hearts. Treatment of mice with bosentan (endothelin‐1 receptor antagonist) normalized the coronary perfusion pressure, demonstrating that the elevated endothelin‐1 release was sufficient to account for the increased coronary perfusion pressure. Pharmacological blockade of K_ATP channels led to elevated endothe‐lin‐1 levels in the coronary effluent of isolated mouse and rat hearts as well as enhanced endothelin‐1 secretion from isolated human coronary endothelial cells. These data are consistent with a role for endothelial K_ATP channels to control the coronary blood flow by modulating the release of the vasoconstrictor, endothe‐lin‐1.–Malester, B., Tong, X.Y., Ghiu, I., Kontogeorgis, A., Gutstein, D. E., Xu, J., Hendricks‐Munoz, K. D., Coetzee, W. A. Transgenic expression of a dominant negative K_ATP channel subunit in the mouse endothelium: effects on coronary flow and endothelin‐1 secretion. FASEB J. 21, 2162–2172 (2007)