The mode of action of venom from the ectoparasitic wasp Nasonia vitripennis in eliciting cell death was examined using an in vitro approach with BTI-TN-5B1-4 cells, and the cell responses were compared to those evoked by the extensively studied wasp toxin mastoparan. Wasp venom increased plasma membrane permeability to Na+, resulting in cellular swelling and death due to oncosis. When ouabain was used to disable Na+, K+-ATPases, the effects of venom were enhanced. Measurements of intracellular calcium using fluo-4 AM revealed a rearrangement and an increase in cytosolic [Ca+2]i within 30 min after exposure of BTI-TN-5B1-4 cells to venom. This venom-mediated increase in Ca+2 was apparently due to mobilization of intracellular stores since the changes occurred in the absence of extracellular Ca+2. Phospholipase C (PLC) inhibitors, neomycin and U-73122, blocked the venom-induced death temporarily (<3h), but by 24h, all venom-treated cells swelled and lysed. Pre-treatment of cells with caffeine or theophylline but not ryanodine attenuated the induction of oncosis by wasp venom. Anti-inflammatory peptide 1 (antiflammin 1) but not bromophenacyl bromide, agents that block phospholipase A2 (PLA2) activity, abolished the responsiveness of BTI-TN-5B1-4 cells to venom. These results suggest that venom initiates cell death by inducing Ca+2 release from intracellular stores probably via phospholipase C and IP3. A possible mode of action for venom from N. vitripennis requiring dual activation of PLC and PLA2 is discussed and compared to the pathways known to be activated by mastoparan.