We have previously demonstrated the vasorelaxant properties of the omega-3 fatty acid, eicosapentaenoic acid (EPA), in normotensive and spontaneously hypertensive rat (SHR) aorta, although the mechanism(s) of action are not fully understood. Because endothelial dysfunction and increased intracellular free calcium concentration ([Ca2+]i) are seen in hypertensive rat aorta, we investigated the potential role of Ca2+ signaling, endothelium and derived factors, and the opening of potassium (K+) channels in EPA-induced relaxation. In the presence of extracellular Ca2+, EPA induced significant relaxations at >10 micromol/L (P<.01) in norepinephrine (NE) (10(-6) mol/L)-contracted aortic rings and at 30 micromol/L (P<.001) in high K+ (80 mmol/L)-contracted aortic rings. In the absence of extracellular Ca2+, EPA (10 to 30 micromol/L) inhibits the tonic component of NE-induced contraction (P<.0001). The relaxant properties of EPA in SHR aorta appear specific to Ca2+ release from an internal storage site associated with NE-induced tonic contraction. Further studies with the use of fura-2 to measure [Ca2+]i in cultured vascular smooth muscle (VSM) cells from SHR aorta indicated that EPA (30 micromol/ L)-pretreatment attenuated angiotensin II (50 nmol/ L)-induced Ca2+ transient by 95%, suggesting that an inhibitory effect on the Ca2+ signaling may underlie EPA-induced relaxation of the vessel preparation. In addition, EPA per se induced an increase in [Ca2+li with a duration of approximately 20 min in VSM cells, and the effect was not altered by removal of extracellular Ca2+. There was no increase in the level of inositol-1,4,5-trisphosphate in response to EPA (30 micromol/L). The actions of EPA are independent of endothelium-derived factors, cyclooxygenase metabolites, and activation of K+ channels since endothelium removal, N(omega)-nitro-L-arginine methyl ester hydrochloride, (L-NAME, 100 micromol/L), indomethacin (10 micromol/L), tetraethylammonium (1 mmol/L), and glibenclamide (10 micromol/L) did not affect EPA-induced vasodilation in NE-precontracted aortic rings. These results suggest that EPA directly modulates intracellular Ca2+ signaling in VSM cells, and that this may contribute to the vasorelaxant effect and, at least in part, the blood pressure-lowering effect of fish oil.