Recently, nitric oxide (NO) signalling pathways have been linked with abnormalities in cardiac repolarisation and cardiac arrhythmias. Studies from isolated heart preparations have demonstrated an antifibrillatory effect following vagus nerve stimulation, which is NO dependent. The cellular and molecular basis for this protective effect is unknown. In this study, cGMP dependent regulation of repolarisation was investigated using BAY 60-2770, a novel NO and haem independent soluble guanylyl cyclase (sGC) activator. Action potentials (APs) and ionic currents were recorded from isolated guinea pig ventricular myocytes at 37oC using the perforated patch clamp technique. APs were stimulated at 2 Hz and the slow and rapid delayed rectifier potassium currents, IKs and IKr were measured as tail currents elicited by a voltage step from +40 mV to -50 mV. Cellular cGMP was quantified by radioimmunoassay and expressed as a fold change relative to basal levels. Values are presented as means ± S.E.M. compared by paired t test. 1 μM BAY 60-2770 increased cellular cGMP by 2.2 ± 0.3 fold (n=8) and by 7.1 ± 0.8 fold (n=7) in the presence of 100 μM IBMX (3-Isobutyl-1-methylxanthine), a non-selective phosphodiesterase (PDE) inhibitor. In vitro assays on purified sGC have shown that ODQ (1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one) potentiates the action of BAY 60-2770. This was corroborated in our experiments, BAY 60-2770 + 10 μM ODQ increased cGMP by 27.9 ± 9.6 fold (n=4). Despite substantial increases in cellular cGMP, changes in times to 90% repolarisation of action potentials (APD90) were modest. BAY 60-2770 modestly, but significantly (p<0.001, n=8) shortened APD90 by 11.3 ms, subsequent addition of ODQ in the presence of BAY 60-2770 caused no significant further APD90 shortening. In contrast, in the presence of IBMX, BAY 60-2770 significantly (p<0.05, n=7) lengthened APD90 by 15.7 ms, suggesting PDE inhibition permits repolarising currents to be inhibited by cGMP dependent signalling pathways. Further experiments showed that IKs, but not IKr, was inhibited by BAY 60-2770, in the presence of IBMX. IBMX enhanced IKs by 77 ± 9 % (p<0.001, n=8). Subsequent addition of BAY 60-2770 reduced the IBMX dependent enhancement of IKs by 68 ± 2 % (p<0.001, n=8). BAY 60-2770 had no significant effect on IKr, either on its own or in the presence of IBMX. Overall, these findings demonstrate the complex interplay between cGMP and cAMP mediated effects on the ion channels regulating cardiac repolarisation. The modest effect of BAY 60-2770 + ODQ on APD90, despite a substantial increase in cellular cGMP, highlights that PDEs limit cGMP accumulation close to ion channels in the sarcolemma, thus compartmentalising cGMP signalling.