Recently, we have reported that excess amounts of nitric oxide (NO) produced by inducible NO synthase are involved in the development of myocardial damage in rats with induced myocarditis. However, there remain many problems to be solved concerning its mechanism of action. In this study, we examined whether NO induces apoptotic cell death in cardiomyocytes. Cultured neonatal rat cardiomyocytes were exposed toS-nitroso-N-acetylpenicillamine (SNAP) and (±)-E-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexeneamine (NOR 3), as NO donors, or 8-bromo-cyclic GMP (cGMP), an analog of cGMP which functions as a second messenger in cells stimulated by NO. DNA fragmentation was confirmed by electron microscopy, by the terminal deoxynucleotidyl transferase-mediated dUTP–biotin nick end labeling (TUNEL) method, and by agarose gel electrophoresis. Exogenously supplied SNAP or NOR 3 induced cardiomyocyte apoptosis in a dose- and time-dependent manner. Cardiomyocytes exposed to SNAP displayed typical features of apoptosis as demonstrated by electron microscopy. Treatment of the cells with 8-bromo-cGMP also induced apoptosis. In cardiomyocytes, SNAP-induced apoptosis was completely blocked by a PKG inhibitor (KT5823) and by a soluble guanylate cyclase inhibitor (ODQ) and was suppressed by hemoglobin and was completely blocked by ZVAD-FMK, a caspase inhibitor. These results show that NO-mediated apoptosis of cardiomyocytes is cGMP dependent and that caspases are involved in this process.