We reported previously that low levels of nitric oxide (NO) induced cell death with properties of apoptosis, including chromatin fragmentation and condensation in undifferentiated PC12 pheochromocytoma cells. The present study demonstrates that cytotoxicity of low concentrations of NO is mediated by inhibition of mitochondrial cytochrome c oxidase and generation of reactive oxygen species (ROS). An NO donor, (±)‐(E)‐4‐ethyl‐2‐[(E)‐hydroxyimino]‐5‐nitro‐3‐hexenamide (NOR3) induced cell death even at low concentrations (10–100 μM), whereas peroxynitrite and a peroxynitrite generator, 3‐(4‐morpholinyl)‐sydnonimine (SIN‐1), did not have a significant effect on cell viability up to a concentration of 0.5 mM. The NOR3‐induced cell death was unaffected by pretreatment with superoxide dismutase (SOD) or its mimetic peroxynitrite scavenger, manganese(III) tetrakis(benzoic acid)porphyrin chloride (Mn‐TBAP), or with uric acid. These findings indicate that peroxynitrite does not contribute to this cell death. Furthermore, neither the release of cytochrome c from mitochondrial membranes, the cleavage of poly‐ADP ribose polymerase (PARP), nor the activation of caspase‐3‐like activities was observed. Inhibitors of PARP, benzamide, and aminobenzamide, had no effect on the NOR3‐induced cell death. In addition, pretreatment with general or selective caspase inhibitors, benzyloxy‐carbonyl‐Val‐Ala‐Asp‐fluoromethylketone (Z‐VAD‐fmk), N‐acetyl‐Asp‐Glu‐Val‐Asp‐aldehyde (Ac‐DEVD‐CHO), and benzyloxycarbonyl‐Asp‐2,6‐dichlorobenzoyloxymethylketone (Z‐Asp‐Ch₂‐DCB) did not prevent NOR3‐induced cell death. Taken together, these findings suggest that cell death induced by NOR3 occurs by a caspase‐independent mechanism. In contrast, we found an early increase in mitochondrial H₂O₂ production during NOR3 exposure using the fluorescent dye 2′,7′‐dichlorofluorescin‐diacetate (DCFH‐DA) and dihydrorohdamine123 (DHR123), and these events were accompanied by strong inhibition of cytochrome c oxidase activity in the cells. Furthermore, we observed that several antioxidants, such as ascorbate, glutathione (GSH), cysteine, tetrahydrobiopterin, and dithiothreitol (DTT), all effectively prevented the NOR3‐induced cell death. NOR3 treatment decreased the level of total intracellular GSH, but did not affect the activities of antioxidant enzymes SOD, GSH‐peroxidase (GPX), and catalase. These results suggest that cell death induced at physiologically low concentrations of NO is mediated by ROS production in mitochondria, most likely resulting from the inhibition of cytochrome c oxidase, with ROS acting as an initiator of caspase‐independent cell death. © 2003 Wiley‐Liss, Inc.