Recently, we showed C-type natriuretic peptide (CNP)-induced negative inotropic (NIR) and positive lusitropic response (LR) in failing rat heart. We wanted to study whether, and if so, how phosphodiesterases (PDEs) regulate CNP-induced cyclic 3',5'-guanosine monophosphate (cGMP) elevation and functional responses. Inotropic and lusitropic responses were measured in left ventricular muscle strips and cyclic nucleotide levels, PDE activity and phospholamban (PLB) and troponin I (TnI) phosphorylation were measured in ventricular cardiomyocytes from Wistar rats with heart failure 6 weeks after myocardial infarction. CNP-mediated increase in global cGMP was mainly regulated by PDE2, as reflected by a marked amplification of the cGMP increase during PDE2 inhibition and by a high PDE2 activity in cardiomyocytes. PDE3 inhibition, on the other hand, caused no significant cGMP increase by CNP. The functional consequences did not correspond to the changes of cGMP. PDE3 inhibition increased the potency of the CNP-induced NIR and LR, while PDE2 inhibition desensitized the CNP-induced NIR, but not LR. A role for PDE2 on the maximal LR and PDE5 on the maximal NIR to CNP was revealed in the presence of PDE3 inhibition. CNP increased PLB phosphorylation about 25- to 30-fold and tended to increase TnI phosphorylation about twofold. As a whole, CNP-induced functional responses were only modestly regulated by PDEs compared to the cAMP-mediated functional responses to β1-adrenoceptor stimulation, which are highly regulated by PDEs. There is a mismatch between the CNP-induced cGMP increase and functional responses. Global cGMP levels are mainly regulated by PDE2 after CNP stimulation, whereas the functional responses are modestly regulated by both PDE2 and PDE3, indicating cGMP compartmentation by PDEs affecting CNP-induced responses in failing hearts.