—Increased production of nitric oxide (NO) after induction of the cytokine-inducible isoform of nitric oxide synthase (iNOS or NOS2) in cardiac myocytes and other parenchymal cells within the heart may in addition to contributing to myocyte contractile dysfunction also contribute to the induction of programmed cell death (apoptosis). To investigate the mechanism(s) by which increased NO production leads to apoptosis, we examined the role of NO in primary cultures of neonatal rat ventricular myocytes (NRVMs) after induction by the cytokines interleukin-1β (IL-1β) and interferon γ (IFNγ) or exposure to the exogenous NO donor S-nitroso-N-acetylcysteine (SNAC) or peroxynitrite (ONOO⁻). Both SNAC (1 mmol/L) and ONOO⁻ (100 μmol/L), but not their respective controls (ie, N-acetylcysteine and pH-inactivated ONOO⁻), induced apoptosis in confluent, serum-starved NRVMs at 48 hours. Similarly, incubation of NRVMs with IL-1β and IFNγ for 48 hours resulted in an increase in iNOS expression, nitrite production, and programmed cell death. Both the cytokine-induced nitrite accumulation and myocyte apoptosis could be completely prevented by the nonselective NOS inhibitor l-nitroarginine (3 mmol/L) or the specific iNOS inhibitor 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (AMT, 100 μmol/L). NO-mediated myocyte apoptosis was not attenuated by the inhibition of soluble guanylyl cyclase with ODQ, nor could apoptosis be induced by the incubation of NRVMs with 1 mmol/L 8-bromo-cGMP, a cell-permeant cGMP analogue. However, NO-mediated apoptosis was significantly attenuated by the superoxide dismutase mimetic and ONOO⁻ scavenger Mn(III)tetrakis (4-benzoic acid) porphyrin (MnTBAP, 100 μmol/L). NO/ONOO⁻-mediated apoptosis was associated with increased expression of Bax with no change in Bcl-2 mRNA abundance. Furthermore, apoptotic cell death was also confirmed in adult rat ventricular myocytes (ARVMs) when grown in heteroculture with IL-1β– and IFNγ-treated rat cardiac microvascular endothelial cells. Therefore, cytokine-induced apoptosis in NRVMs and ARVMs is mediated by iNOS induction, ONOO⁻, and associated with an increase in Bax levels.