ABSTRACT.    In previous investigations we have shown that climacostol induces programmed cell death (PCD) in human epithelial carcinoma cell line (A431) and human promyelocytic leukemia (HL60) cells. The experimental data collected in this study demonstrate that climacostol also induces early intracellular reactive oxygen species (ROS) generation in HL60 cells, which in turn affects cell survival. In particular, our aim was to evaluate the effects of the antioxidant N-acetyl-L-cysteine (NAC) on ROS production, cytotoxicity and caspase activity in climacostol-treated HL60 cells. For this purpose, cells were treated with increasing concentrations of climacostol (0-10 μg/ml) in the absence and in the presence of 5 mM NAC. We observed that: (1), NAC can effectively inhibit 50-55% ROS generation in climacostol-treated cells; (2), NAC significantly reduced the cytotoxic potential of climacostol, which in lactate dehydrogenase activity (LDH) cytotoxicity assay showed values decreasing from 42.16%-81.4% to 19.1%-45.2%, in the range of toxin concentration (1.25-10 μg/ml); (3), the caspase activity was significantly reduced (45-50%) by pre-incubation with NAC of HL60 cells exposed to climacostol. In conclusion, our results suggest that: (1) in HL60 cells the viability decrease and PCD induced by climacostol are mediated by ROS; (2) at least the mitochondrial death pathway is triggered by the toxin; (3) the antioxidants NAC can protect HL60 cells against the effect of climacostol by reducing the ROS levels.