Nitric oxide (NO) signalling has been linked with the regulation of cardiac repolarisation. Abnormalities in cardiac repolarisation are associated with an increased risk of arrhythmia and sudden cardiac death. Studies from isolated heart preparations with intact innervation have demonstrated a clear antifibrillatory effect following vagus nerve stimulation, which is dependent on neuronally released NO. The cellular and molecular basis for this protective effect is unknown. In this study, NO regulation of repolarisation via activation of cGMP dependent pathways was investigated using a novel NO and haem-independent activator of soluble guanylyl cyclase (sGC) – BAY 60-2770. All experiments were performed on acutely isolated adult ventricular guinea pig myocytes maintained in Tyrode at 35-37 °C. The perforated patch current clamp technique was used to record action potentials (AP) and times to 90% repolarisation (APD90) were determined in cells paced at 2 Hz. Cellular cGMP was quantified by radio-immunoassay and expressed as a fold change relative to basal levels. Data are presented as mean ± SEM. 1 µM BAY 60-2770 increased cellular cGMP by just 2.1 ± 0.2 (n = 5) fold on its own and by 7.9 ± 0.7 (n = 4) fold 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, and this was corroborated in our experiments by 1 µM BAY 60-2770 + 10 µM ODQ causing a dramatic 38.7 (n = 2) fold increase of cGMP. Despite these substantial increases in cellular cGMP levels with sGC activation, APD90 changes were relatively modest except when PDEs were inhibited. Thus, 1 µM BAY 60-2770 alone resulted in a small, but statistically significant (p < 0.001) shortening of APD90 from 223.3 ± 8.1 ms (n = 6) in basal conditions to 213.0 ± 7.4 ms (n = 6), and adding ODQ to BAY 60-2770 caused no significant further APD90 shortening. In contrast, when cGMP hydrolysis was blocked by IBMX (100 µM) and protein kinase A was inhibited by H89 (3 µM), BAY 60-2770 caused a pronounced lengthening of APD90 of 19.5 ± 5.6 ms (p < 0.01, n = 8). This lengthening occurred in the late rather than early stages of the AP, supporting our preliminary findings that delayed rectifier potassium currents are modulated by these pathways. Overall, our results demonstrate the complex interplay between cGMP and cAMP mediated effects on the ion channels regulating cardiac repolarisation. The relative lack of effect of BAY 60-2770 + ODQ on APD90, despite large changes in cellular levels of cGMP, provide further evidence that PDEs limit cGMP accumulation close to ion channels in the sarcolemma and demonstrate the functional importance of compartmentalisation of cGMP.