20-HETE is a potent constrictor of small blood vessels and has been suggested to play a crucial role in the generation of myogenic tone and the development of hypertension. In the present study, we investigated the mechanisms by which exogenously applied 20-HETE modulates vascular tone in small porcine coronary arteries. In organ chamber experiments, 20-HETE elicited a concentration-dependent contraction of small porcine coronary artery rings that was partially inhibited by the cyclooxygenase inhibitor diclofenac, the thromboxane and endoperoxide receptor antagonist SQ29548, and the thromboxane A2 synthase inhibitor furegrelate. Removal of endothelium attenuated the response to 20-HETE, whereas preconstriction of endothelium-denuded vessels to 25% of the maximum response with KCl markedly enhanced the response to 20-HETE. This 20-HETE-induced contraction was not associated with a significant increase in the intracellular concentration of Ca2+. 20-HETE-induced contraction was also observed in beta-escin-permeabilized arteries precontracted with a submaximal concentration of Ca2+ and was abolished by the Rho-kinase inhibitor Y27632, but was insensitive to the PKC inhibitor RO 31-8220. 20-HETE elicited the phosphorylation of the myosin light chain (MLC20) in coronary artery rings, an effect that was sensitive to Y27632 and mimicked by the thromboxane analog U46619. These data suggest that in small porcine coronary arteries, 20-HETE can induce contraction by 2 mechanisms, one endothelium-dependent involving the cyclooxygenase-dependent generation of vasoconstrictor prostanoids, and the other endothelium-independent. The latter response is associated with the activation of Rho-kinase, phosphorylation of MLC20, and sensitization of the contractile apparatus to Ca2+.