Xestospongins (Xe's) A, C, D, araguspongine B, and demethylxestospongin B, a group of macrocyclic bis-1-oxaquinolizidines isolated from the Australian sponge, Xestospongia species, are shown to be potent blockers of IP₃-mediated Ca²⁺ release from endoplasmic reticulum vesicles of rabbit cerebellum. XeC blocks IP₃-induced Ca²⁺ release (IC₅₀ = 358 nM) without interacting with the IP₃-binding site, suggesting a mechanism that is independent of the IP₃ effector site. Analysis of Pheochormocytoma cells and primary astrocytes loaded with Ca²⁺-sensitive dye reveals that XeC selectively blocks bradykinin- and carbamylcholine-induced Ca²⁺ efflux from endoplasmic reticulum stores. Xe's represent a new class of potent, membrane permeable IP₃ receptor blockers exhibiting a high selectivity over ryanodine receptors. Xe's are a valuable tool for investigating the structure and function of IP₃ receptors and Ca²⁺ signaling in neuronal and nonneuronal cells.