Background and Methods: Different K⁺ currents have been implicated in the myocardial action potential repolarization including the I_Kr. ERG1 asubunits, identified as the molecular correlate of I_Kr, have been shown to form heteromultimeric channels in the heart and their activity is modulated by a complex interplay of signal transduction events. Using electrophysiological techniques, we examined the effects of the cGMP-analogue 8-Br-cGMP on the rat and guinea-pig papillary action potential duration (APD) and on the biophysical properties of homo- and heteromeric ERG1 channels expressed in HEK-293 cells. Results: 8-Br-cGMP reduced papillary APD by about 30%, but prolonged APD by about 25% after pharmacological inhibition of L-type Ca²⁺ currents and I_Ks. The prolongation was completely abolished by prior application of the hERG channel blocker E-4031 or the protein kinase G (PKG) inhibitor Rp-8-Br-cGMPS. Expression analysis revealed the presence of both ERG1a and -1b subunits in rat papillary muscle. Both 8-Br-cGMP and ANP inhibited heterologously expressed ERG1b and even stronger ERG1a/1b channels, whereas ERG1a channels remained unaffected. The inhibitory 8-Br-cGMP effects were PKG-dependent and involved a profound current reduction accompanied by a leftward-shift in the voltage dependence of steady-state inactivation and an acceleration of inactivation and deactivation kinetics. Cardiac AP clamp recordings confirmed a considerable reduction of ERG1a/1b currents by 8-Br-cGMP. Conclusion: These findings provide first evidence that through heteromeric assembly ERG1 channels become a critical target of cGMP-PKG signaling linking cGMP accumulation to cardiac I_Kr modulation.