The recently described cAMP sensor, Exchange protein directly activated by cAMP (Epac), has been implicated in distinct cAMP-dependent, protein kinase A-independent cellular signalling pathways (Bos JL, 2006). We investigated effects of Epac activation in catecholamine-induced ventricular arrhythmogenesis. In contrast to control findings (n = 20), monophasic action potentials showed spontaneous triggered activity in 2 out of 10 intrinsically beating and 5 out of 20 extrinsically-paced Langendorff-perfused murine hearts perfused with the specific Epac activator 8-pCPT-2’-O-Me-cAMP (8-CPT, 1 μM) (Christensen AE et al. 2003). During steady extrinsic pacing at 8 Hz, 3 out of 20 such hearts showed spontaneous ventricular tachycardia (VT). Programmed electrical stimulation provoked VT in 10 of 20 similarly treated hearts (P < 0.001; n = 20, Fisher’s Exact Test). However, no statistically significant changes (P > 0.05, ANOVA) in left ventricular epicardial (40.7 ± 1.2 versus 44.0 ± 1.7 ms; n = 10), or endocardial action potential durations (APD₉₀) (51.8 ± 2.3 versus 51.9 ± 2.2 ms; n = 10), transmural (ΔAPD₉₀) (11.1 ± 2.6 versus 7.9 ± 2.8 ms; n = 10) or apico-basal gradients of repolarization, ventricular effective refractory periods (29.1 ± 1.7 versus 31.2 ± 2.4 ms in control and 8-CPT-treated hearts, respectively; n = 10) and APD₉₀ restitution characteristics accompanied these arrhythmogenic effects. However, fluo-3 fluorescence imaging of cytosolic Ca²⁺ demonstrated alterations in Ca²⁺ homeostasis in the form of increased Ca²⁺ wave generation in both paced and resting isolated 8-CPT-treated ventricular myocytes. An independent method of Epac activation that applied 100 nM isoproterenol to stimulate beta-adrenoreceptors in parallel with protein kinase A inhibition by 2 μM H-89, was also arrhythmogenic in the whole heart and similarly altered cytosolic Ca²⁺ homeostasis. The Epac-dependent effects at both the whole heart and cellular levels were reduced by inhibition of Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) with 1 μM KN-93. These findings associate VT in an intact cardiac preparation with altered cellular Ca²⁺ homeostasis and Epac activation through a CaMKII-dependent mechanism for the first time, in the absence of the altered repolarization gradients previously implicated in re-entrant arrhythmogenesis (Killeen MJ et al. 2007; Thomas G et al. 2007).