Venom from the ectoparasitic wasp Nasonia vitripennis mobilizes calcium from intracellular stores prior to the onset of oncotic cell death. We investigated the potential source of the intracellular using an in vitro system and crude wasp venom. Venom appeared to initially trigger a rapid and sharp decline in mitochondrial membrane potential that preceded a rise in cytosolic intracellular calcium. Agents that block calcium release channels associated with IP3, RyR and Ca+2-ATPase receptors on endoplasmic reticulum neither attenuated or augmented the effects of wasp venom. However, verapamil abolished venom-mediated death. We propose that venom mobilizes calcium from intracellular stores by stimulating unregulated efflux of calcium from mitochondria into the cytosol as a consequence of a loss of mitochondrial membrane integrity, and subesequently triggers further release of calcium from endoplasmic reticulum via the action of cAMP modulating NAADP-sensitive calcium stores.