C-type natriuretic peptide (CNP) and endothelin-1 are paracrine peptides with opposing effects on cardiac myocyte contraction and intracellular cGMP production. Elevated levels of both endothelin-1 and CNP are found in patients with congestive heart failure. These factors may be related to positive and negative regulation of cell apoptosis in the failing heart. To evaluate the effect of CNP and endothelin-1 on apoptosis of cardiac myocytes and the possible mechanisms involved, primary cardiac myocytes were prepared from neonatal Sabra rats. Cardiomyocyte apoptosis was evaluated by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and Annexin V in situ staining. The TUNEL method was used to measure the apoptotic index. CNP and the cGMP derivative, 8-br-cGMP, induced apoptosis of cardiac myocytes. CNP-induced apoptosis could be blocked by HS 142-1 (a mixture of 20–30 kinds of linear β-1, 6-glucan esterified by capronic acid, an antagonist of type A and B natriuretic peptide receptors), and KT 5823 (C₂₉H₂₅N₃O₅, the inhibitor of cGMP-dependent protein kinase). α-Difluoromethylornithine (DFMO), the irreversible inhibitor of ornithine decarboxylase, also induced apoptosis to a similar extent. CNP and 8-br-cGMP caused a marked reduction of intracellular ornithine decarboxylase expression, as determined by Western blot analysis and immunohistochemical assay. Preincubation with endothelin-1 attenuated CNP- and 8-br-cGMP-induced cardiomyocyte apoptosis. Endothelin-1 also antagonized the CNP- and 8-br-cGMP-induced reduction of intracellular ornithine decarboxylase expression. These results suggest that CNP has a proapoptotic effect on neonatal rat cardiac myocytes. The effect is mediated via natriuretic peptide receptors and is due to an elevation of intracellular cGMP, which reduces the expression of intracellular ornithine decarboxylase and probably the production of polyamines. Endothelin-1 protects cardiac myocytes against CNP-induced apoptosis by influencing the cGMP-dependent pathway, and this effect is probably mediated through both a reduction of cGMP and antagonism of the CNP-induced reduction of intracellular ornithine decarboxylase expression.