β-adrenergic receptor (β-AR) stimulation increases cardiac function by increasing cAMP levels and activating protein kinase A (PKA). PKA enhances Ca2+-induced Ca2+-release by phosphorylating L-type Ca2+ channels, ryanodine receptors and phospholamban which are also targets of the Ca2+/calmodulin kinase II (CaMKII). Any disregulation in the β-adrenergic pathway can lead to cardiac arrhythmias. Multiple cyclic nucleotide phosphodiesterases (PDEs) regulate local concentrations of cAMP, among which the PDE4 family plays an important role. The aim of this study is to investigate the proarrythmic effects of PDE4 inhibition and evaluate the relative contribution of PKA and CaMKII in this mechanism. Action potentials (AP) were recorded at a frequency of 1 Hz in isolated adult Wistar rat ventricular myocytes using the patch clamp technique. Application of the non selective β-AR agonist Isoprenaline (Iso 1 nM) induced delayed afterdepolarizations (DADs) recorded during 10 s after the cessation of electrical stimulation which were potentiated by the selective PDE4 inhibitor Ro20-1724 (1.5±0.3 DADs/10s, n=18 with Iso alone, p<0.01 versus control; and 2.9±0.5 DADs/10s with Iso+10 µM Ro20-1724, p<0.01 versus Iso alone, values are means±S.E.M. compared by one-way ANOVA with Tukey post hoc test). These DADs correlated with spontaneous calcium release events (SCRs) recorded with an IonOptix® system in myocytes loaded with Fura-2AM (5 µM). The SCRs appeared upon Iso application (1 nM) and their incidence increased from 1.6±0.4 SCRs/10s (n=21) in Iso alone to 4.3±0.5 SCRs/10s (n=25; p<0.001) when PDE4 was concomitantly inhibited. The SCRs observed in the presence of Iso+Ro were abolished in cells preincubated with the PKA inhibitor H-89 (10 µM) (n=6; p<0.001) and their occurrence diminished by 40% when CaMKII was inhibited by 10 µM KN-93 (n=23; p<0.05). The inactive KN92 (10 µM) compound had no effect on SCRs (n=13). These results show that upon β-AR stimulation, PDE4 inhibition promotes DADs due to SCRs through both PKA and CaMKII activation.