Serum albumin protects against cell death elicited by various cytotoxic agents; however, conflicting views on the protective mechanism still remain. Hence, we have studied the ability of serum albumin to prevent apoptosis of human neuroblastoma SH-SY 5 Y cells elicited by four compounds known to release Ca(2+) from the endoplasmic reticulum, i.e. dotarizine, flunarizine, thapsigargin and cyclopiazonic acid. Spontaneous basal apoptosis, after 24 h incubation in Dulbecco's Modified Eagle Medium (DMEM) containing 10% serum, was 5%. Dotarizine (30--50 microM) enhanced basal apoptosis to 18--43%, flunarizine (30--50 microM) to 15%, thapsigargin (1--10 microM) to 21--35%, and cyclopiazonic acid (100 microM) to 10%. Serum deprivation augmented basal apoptosis to 20%. Under serum-free medium, 30 microM dotarizine or flunarizine drastically enhanced apoptosis to 63% and 68%, respectively; the increase was milder with 1 microM thapsigargin (37%) and 30 microM cyclopiazonic acid (27%). In serum-free medium, albumin (29 or 49 mg/ml) fully prevented the apoptotic effects of dotarizine, flunarizine and cyclopiazonic acid. The four compounds increased the cytosolic Ca(2+) concentration ([Ca(2+)](c)) in fluo-4 loaded cells; such increase developed slowly to reach a plateau after several minutes, followed by a slow decline. Albumin did not modify the kinetic parameters of such increase. In the absence of serum, dotarizine, flunarizine, thapsigargin, and cyclopiazonic acid caused mitochondrial depolarization in tetramethylrhodamine ethyl ester (TMRE)-loaded cells; depolarization was inhibited by cytoprotective concentrations of albumin. These results suggest that albumin protects cells from entering into apoptosis by preventing mitochondrial depolarization. They also suggest that inhibition of mitochondrial depolarization might become a target to develop new anti-apoptotic compounds with therapeutic neuroprotective potential in stroke, Alzheimer's disease, and other neurodegenerative diseases.