Multiple cyclic nucleotide phosphodiesterases (PDEs) belonging to four families (PDE1 to PDE4) hydrolyze cAMP in cardiac cells, but the functional significance of this diversity is not well understood. The goal of this study was to characterize the involvement of different PDEs in excitation-contraction coupling in cardiomyocytes. For this, sarcomere shortening and Ca2+ transients were recorded simultaneously in rat ventricular myocytes field stimulated at 0.5 Hz with an IonOptix system. Selective inhibition of PDE2 with Bay 60-7550 (Bay, 100nM) or PDE4 with Ro-201724 (Ro, 10μM) had no effect on basal cell contraction, whereas selective inhibition of PDE3 with cilostamide (Cil, 1μM) or β-adrenergic stimulation with isoprenaline (Iso, 1nM) increased myocyte shortening. Inhibition of PDE4 potentiated the response to Cil and Iso, showing that PDE4 becomes important when cAMP is prestimulated. Similar results were obtained on Ca2+ transients. cAMP measurements by FRET in beating cardiomyocytes indicate that Iso strongly increases cAMP levels. Effects of selective PDE inhibitors are under investigation. These results show that PDE2, PDE3 and PDE4 differentially regulate excitation-contraction coupling in cardiomyocytes.