Inhibiting Ca²⁺ uptake by the sarcoendoplasmic reticular Ca²⁺-ATPase pump (SERCA) causes release of Ca²⁺ from the endoplasmic reticulum (ER), increased cytosolic Ca²⁺ ([Ca²⁺]_cyt) and depletion of ER Ca²⁺ stores. These studies were designed to test the effects of SERCA inhibition on neuronal viability, using as a model the human neuroblastoma cell line, SH-SY5Y. Continuous exposure to the SERCA inhibitor thapsigargin (TG) decreased SH-SY5Y viability to <30% after 48 h exposure, and produced DNA laddering. Two other SERCA inhibitors, BHQ and cyclopiazonic acid CPA, were similarly toxic, although at 1000-fold higher concentrations. BHQ and CPA toxicity was prevented by removing drug within several hours, whereas TG toxicity was essentially irreversible. All three SERCA inhibitors caused an increase in [Ca²⁺]_cyt that was partially blocked by the ryanodine receptor inhibitors, dantrolene and DHBP. Pretreatment with 40 μM dantrolene gave substantial protection against TG- or BHQ-induced cell death but it did not inhibit death from staurosporine, which does not cause release of ER Ca²⁺. DHBP (20–100 μM) also gave partial protection against TG toxicity, as did ruthenium red (2 μM), but not ryanodine (10 μM). Inhibition of capacitative Ca²⁺ entry with EGTA or LaCl₃ or low extracellular Ca²⁺, or chelation of [Ca²⁺]_cyt with BAPTA-AM, failed to inhibit TG toxicity, although they prevented increases in [Ca²⁺]_cyt caused by TG. Taken together, these data suggest that toxicity caused by SERCA inhibition in SH-SY5Y cells is caused by ER Ca²⁺ depletion, which triggers an apparent apoptotic pathway.