Natriuretic peptides (NPs) are used as biomarkers in heart failure (HF) as they increase with severity of the disease. C-type natriuretic peptide (CNP) and brain natriuretic peptide (BNP) activate NPR-B and NPR-A receptors, respectively. Earlier studies have shown that CNP elicits a direct negative inotropic response (NIR) and a positive lusitropic response (LR) through the cGMP - protein kinase G pathway. In this study we investigated cGMP increase and functional responses to CNP and BNP and the regulation by phosphodiesterases (PDEs) in isolated ventricular cardiomyocytes and muscle strips from Wistar rats with HF. CNP and BNP both increased cGMP levels, but only CNP caused functional responses and increased PLB and TnI phosporylation, the Ca2+ transient magnitude and the Ca2+ extrusion rate constant. Preincubation with BNP did not affect the CNP-induced NIR, however it increased the CNP-induced LR. cGMP measurements indicated that NPR-A and NPR-B stimulation involved different cGMP compartments. Both BNP- and CNP-induced cGMP increase is regulated by PDE2, 3 and 5 but a NIR to BNP was not revealed, even in an attempt to abolish compartmentation by the presence of combined PDE2, 3 and 5 inhibition. In conclusion, there is a strong functional compartmentation of the cGMP signal indicating different roles of BNP and CNP in the pathophysiology of HF.