We have recently reported that mastoparan, a peptide toxin isolated from wasp venom, induces apoptosis in cultured cerebellar granule neurons that can be blocked by cholera toxin, an activator of Gs. Measurements of intracellular free calcium concentration ([Ca2+]i) reveal that mastoparan induces a dramatic elevation of [Ca2+]i that is frequently followed by enhanced leakage of fura-2 out of the neurons, suggesting that this rise in [Ca2+]i may be due to a more generalized change in membrane permeability. However, the mastoparan-induced initial elevation of [Ca2+]i is maintained in the absence of extracellular Ca2+, suggesting that the rise of [Ca2+]i is from intracellular stores. This conclusion is supported by the observation that depletion of [Ca2+]i stores by pretreatment with either caffeine or thapsigargin attenuates both the rise in [Ca2+]i and cell death induced by mastoparan. Phospholipase C (PLC) inhibitors, neomycin and U73122 block mastoparan-induced increases of [Ca2+]i and protect against neuronal death. Pretreatment with cholera toxin, but not pertussis toxin, reduced the mastoparan-induced rise in [Ca2+]i. Taken together, our data suggest that mastoparan initiates cell death in cerebellar granule neurons by inducing Ca2+ release from intracellular stores, probably via activation of PLC and IP3. A secondary or parallel process results in disruption of plasma membrane integrity and may be ultimately responsible for the death of these neurons by mastoparan.