Hydrazine (HzN) is an aircraft fuel and propellant used by the U.S. Air Force. The current study was undertaken to evaluate the acute toxicity of HzN in primary rat hepatocytes in vitro with reference to oxidative stress. The effects of short-term exposure (4 h) of hepatocytes to HzN were investigated with reference to viability, mitochondrial function, and biomarkers of oxidative stress. The viability data showed an increase in lactate dehydrogenase leakage and a decrease in mitochondrial activity with increasing concentration of HzN. The results of studies of oxidative stress biomarkers showed a depletion of reduced glutathione (GSH) and an increase in oxidized GSH, increased reactive oxygen species generation, lipid peroxidation, and reduced catalase activity. Furthermore, depletion of GSH and catalase activity in hepatocytes by buthionine sulfoximine and 3-amino triazole, respectively, prior to exposure to HzN, increased its toxicity. The results suggest that acute HzN-induced cytotoxicity in rat hepatocytes is likely to be mediated through oxidative stress.