Vasodilator-induced elevation of intracellular cyclic AMP (cAMP) is a central mechanism governing arterial relaxation [1]. The vasculature expresses three distinct cAMP effectors: cAMP-dependent protein kinase (PKA), cyclic nucleotide-gated ion channels and the more recently discovered exchange protein directly activated by cAMP (Epac) [2]. The mechanisms by which cAMP induces vasorelaxation are thus likely to be complex and diverse. Here we investigate the role of Epac in mediating vasorelaxation in rat mesenteric arteries. In myography experiments, the Epac-selective cAMP analogue 8-pCPT-2′-O-Me-cAMP-AM (5µM, subsequently referred to as 8-pCPT-AM) elicited a 77.6 ± 7.1% relaxation of phenylephrine-contracted arteries over a 5 minute period (mean ± SEM; n=6). 8-pCPT-AM induced only a 16.7 ± 2.4% relaxation in arteries pre-contracted with high extracellular K+ over the same time period (n=10), suggesting that Epac’s relaxant effect relies upon vascular cell hyperpolarization. This involves Ca2+-sensitive, large-conductance K+ (BKCa) channel opening since iberiotoxin (100nM) significantly reduced the ability of 8-pCPT-AM to reverse phenylephrine-induced contraction (arteries relaxed by only 35.0 ± 8.5% over a 5 minute exposure to 8-pCPT-AM, n=5; p<0.05 Student’s t-test). 8-pCPT-AM increased Ca2+ spark frequency in Fluo-4-AM-loaded mesenteric myocytes from 0.045 ± 0.008 to 0.103 ± 0.022 sparks/s/µm (p<0.05) and reversibly increased both the frequency (0.94 ± 0.25 to 2.30 ± 0.72s-1) and amplitude (23.9 ± 3.3 to 35.8 ± 7.7pA) of spontaneous transient outward currents (STOCs) recorded in isolated mesenteric myocytes (n=7; p<0.05). 8-pCPT-AM-activated STOCs were sensitive to iberiotoxin (100nM) and to ryanodine (30µM). Current clamp recordings of isolated myocytes showed a 7.4 ± 0.9mV (n=4) hyperpolarization in response to 8-pCPT-AM. Endothelial disruption suppressed 8-pCPT-AM-mediated relaxation in phenylephrine-contracted arteries (24.8 ± 4.9% relaxation after 5 minutes exposure, n=5; p<0.05), as did apamin and TRAM-34, blockers of Ca2+-sensitive, small- and intermediate-conductance K+ (SKCa and IKCa) channels, respectively, and NG-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthase (NOS). In Fluo-4-AM-loaded mesenteric endothelial cells, 8-pCPT-AM induced a sustained increase in cytosolic Ca2+ in 22 out of 47 cells. Our data suggest that Epac hyperpolarizes smooth muscle by 1) increasing localized Ca2+ release from ryanodine receptors (Ca2+ sparks) to activate BKCa channels, and 2) endothelial-dependent mechanisms involving the activation of SKCa/IKCa channels and NOS. Epac-mediated smooth muscle hyperpolarization will limit Ca2+ entry via voltage-sensitive Ca2+ channels and represents a novel mechanism of arterial relaxation.