<p>Sympathetic activation robustly increases the slow delayed rectifier K⁺ current (I_Ks) in the mammalian ventricular myocardium, however, exact downstream pathways involved in the β-adrenergic regulation of the current are not fully elucidated yet. This study examined the Ca²⁺ sensitivity of I_Ks and the contribution of the protein kinase A (PKA) and the calcium/calmodulin kinase II (CaMKII) pathways in regulating I_Ks in isolated canine ventricular myocytes. Experiments were carried out under ß-adrenergic receptor activation (10&#xa0;nM isoproterenol) and under baseline conditions (without isoproterenol). I_Ks was measured as an HMR-1556 sensitive current with the action potential voltage clamp technique under the physiological intracellular calcium homeostasis of the cells. Reducing intracellular Ca²⁺ concentration ([Ca²⁺]_i) with 1&#xa0;µM nisoldipine decreased the peak and mid-plateau densities of I_Ks, reduced the current integral, and increased the time-to-peak value. In contrast, ß-adrenergic receptor activation by isoproterenol resulted in larger I_Ks densities and integral, and shorter time-to-peak value. These effects of isoproterenol on I_Ks were significantly smaller when the CaMKII inhibitor 1&#xa0;µM KN-93 was present in the cells, but the PKA inhibitor 3&#xa0;µM H-89 did not exert such effect. Importantly, all effects of isoproterenol on I_Ks have fully developed even in the presence of 1&#xa0;µM nisoldipine. Under baseline conditions the mid-plateau density of I_Ks, was significantly smaller in the presence of KN-93, H-89 or nisoldipine, while peak I_Ks density and the current integral were significantly smaller only in nisoldipine. In conclusion, many different signaling pathways are involved in regulating I_Ks. Under baseline conditions the regulation is strongly [Ca²⁺]_i-dependent, with PKA and CaM-CaMKII involved, whereas during ß-adrenergic stimulation it is [Ca²⁺]_i-independent and supposes a pivotal role of EPAC-mediated activation of CaMKII.</p>