The current study was undertaken to investigate the role of apoptosis in hydrazine induced hepatotoxicity. Hepatocytes were exposed to hydrazinium nitrate (HzN) at two doses (50 and 75 mM) for 2 h then placed in fresh HzN-free media and cultured for an additional 24 h. Post-exposure, cell viability was evaluated at several time points by lactate dehydrogenase (LDH) leakage and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction. Markers of apoptosis (mitochondrial membrane potential, annexin binding, DNA fragmentation, caspase activation, and cytochrome c release) were measured 24 h post-exposure. The viability data showed time dependent increase in LDH leakage at 75 mM of HzN, with only a slight increase at 50 mM. MTT reduction showed a decrease in mitochondrial activity at both doses immediately after the 2 h continuous exposure. However, MTT reduction returned to normal at 50 mM while at 75 mM, MTT reduction initially recovered but then deteriorated to approximately 50% of controls at 24 h post-exposure. Based on viability data, exposure to 50 mM HzN for 2 h is a marginally toxic dose while 75 mM is a significantly toxic dose. The results for apoptosis biomarkers showed a reduction in mitochondrial membrane potential, an increase in annexin binding, an increase in total caspase activity, moderate activation of caspase-3, and release of cytochrome c. However, the appearance of DNA fragmentation in HzN exposed cells was very low compared to positive controls (cadmium and cyclosporine). The possibility that HzN induces apoptosis without the involvement of DNA fragmentation can not be ruled out. The present results, overall, suggest that apoptosis may be a contributing factor in acute HzN toxicity.