Intracellular calcium ions regulate the structure and functions of cytoskeletal proteins. On the other hand, recent studies have shown that the cytoskeleton, and actin filaments in particular, can modulate calcium influx through plasma membrane ligand- and voltage-gated channels. We now report that calcium release from inositol trisphosphate (IP3) and ryanodine-sensitive endoplasmic reticulum (ER) stores is modulated by polymerization and depolymerization of actin filaments in cultured hippocampal neurons. Depolymerization of actin filaments with cytochalasin D attenuates calcium release induced by carbamylcholine (CCh; a muscarinic agonist for IP3 pathway), caffeine (a ryanodine receptor agonist) and thapsigargin (an inhibitor of the ER calcium- ATPase) in both the presence and absence of extracellular calcium. Conversely, the actin polymerizing agent jasplakinolide potentiates calcium release induced by CCh, caffeine and thapsigargin. Cytochalasin D attenuated, while jasplakinolide augmented, thapsigargin-induced JNK activation and neuronal cell death. Our data show that the actin cytoskeleton regulates ER calcium release, suggesting roles for actin in the various physiological and pathological processes that involve calcium release.