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Oxidative injury is believed to be a major factor in the pathogenesis of a variety of neurodegenerative diseases. Additionally, the mode of cell death in oxidant-stressed cells can vary. The present study was conducted to evaluate the use of a primary neuronal cell-based bioassay in which different modes of oxidant-induced cell death could be studied and in which putative neuroprotective agents could be screened. Addition of 50 microM H(2)O(2) to primary cortical neuronal cultures for 1 h under normal ATP conditions resulted in approximately 40% cell death, almost exclusively of an apoptotic nature. In this condition, cell death was effectively blocked by GM1 ganglioside, the semi-synthetic ganglioside derivative LIGA20, the dopamine receptor agonist pramipexole (PPX) and the caspase inhibitor Z-VAD-FMK but not by the poly (ADP-ribose) polymerase (PARP) inhibitor 3-aminobenzamide (3-AB). Pretreatment of cells with 0.01 microM oligomycin for 45 min prior to addition of 50 microM H(2)O(2) caused significant ATP depletion and approximately the same amount of cell death as H(2)O(2) alone. However, under these conditions, cell death was primarily non-apoptotic in nature and GM1, LIGA20 and Z-VAD-FMK had no protective effects. In contrast, AB and PPX effectively blocked cell death. These results suggest that cellular ATP plays a critical role in determining the mode of cell death in primary neurons and that these types of in vitro models may provide a useful system for screening putative neuroprotective agents.