Nitric oxide (NO) can be either cytoprotective or cytotoxic in hepatocytes, depending on conditions within the cell. We hypothesized that redox status is a determinant of NO effects on cell viability. To cause the disturbance of redox homeostasis in the hepatocytes, cells were treated with the following glutathione (GSH) depleting agents: (1) chronic depletion by 18 hours pretreatment with buthionine sulfoximine (BSO), which depletes GSH by blocking its biosynthesis; and (2) acute depletion by 1 hour pretreatment with diethyl maleate (DEM), which conjugates GSH by the GSH-S-transferase catalyzed reaction. S-nitroso-N-acetyl-D,L-penicillamine (SNAP), a NO donor, was added after removal of GSH-depleting agents. Individual treatment with either SNAP or GSH depletion did not appreciably affect viability. A significant increase of cytotoxicity in hepatocytes was observed with the combination of a concentration and time course regimen of SNAP and GSH depletion. SNAP treatment of GSH-depleted hepatocytes led to an increase in LDH release and oxidative stress, disruption of mitochondrial membrane potential, the presence of nitrotyrosine (an indicator of peroxynitrite (ONOO) generation), and a decrease in adenosine triphosphate (ATP) content. The interference of mitochondrial respiratory enzymes, especially with the combination treatments, indicated different levels of disturbance of electron transfer, superoxide generation, and ATP production. Other commonly used NO donors were found to exhibit lower and slower toxicity in the setting of GSH depletion than that evident with SNAP. In conclusion, the disruption of cellular redox homeostasis by GSH depletion leads hepatocytes to be more susceptible to NO (especially S-nitrosothiols) and subsequent necrotic cell death. (HEPATOLOGY 2005;42:598–607.)