The effect of NPC-14686, a potential anti-inflammatory drug, on cytosolic free Ca 2+ levels ([Ca 2+ ] i ) and growth in PC3 human prostate cancer cells was examined by using fura-2 as a fluorescent Ca 2+ indicator and WST-1 as a fluorescent growth dye. NPC-14686 at concentrations above 10 μM increased [Ca 2+ ] i in a concentration-dependent manner with an EC 50 value of 100 μM. NPC-14686-induced Ca 2+ influx was confirmed by Mn 2+ quench of fura-2 fluorescence. The Ca 2+ signal was also reduced by removing extracellular Ca 2+ . Pretreatment with 1 μM thapsigargin (an endoplasmic reticulum Ca 2+ pump inhibitor) to deplete the endoplasmic reticulum Ca 2+ nearly abolished 200 μM NPC-14686-induced Ca 2+ release; and conversely pretreatment with NPC-14686 completely inhibited thapsigargin-induced Ca 2+ release. The Ca 2+ release induced by 200 μM NPC-14686 was not affected by inhibiting phospholipase C with 2 μM U73122. Overnight treatment with 1–500 μM NPC-14686 decreased cell viability in a concentration-dependent manner. These findings suggest that in human PC3 prostate cancer cells, NPC-14686 increases [Ca 2+ ] i by evoking extracellular Ca 2+ influx and releasing intracellular Ca 2+ from the endoplasmic reticulum via a phospholiase C-independent manner. NPC-14686 may be cytotoxic to prostate cancer cells.