The effect of nortriptyline, a tricyclic antidepressant, on Ca2+ regulation and viability in human prostate cancer cells (PC3) is unclear. The present study examined whether nortriptyline altered basal [Ca2+]i levels in suspended PC3 cells using fura-2 as a Ca2+-sensitive fluorescent probe. Nortriptyline (50–500 µM) increased [Ca2+]i in a concentration-dependent fashion. The Ca2+ signal was partially reduced by removing extracellular Ca2+, indicating that Ca2+ entry and release both contributed to the [Ca2+]i rise. Nortriptyline induced Mn2+ influx, leading to quench of fura-2 fluorescence, suggesting Ca2+ influx. This Ca2+ influx was inhibited by activation of protein kinase C, but not by inhibition of L-type Ca2+ channels. In Ca2+-free medium, pretreatment with the endoplasmic reticulum Ca2+ pump inhibitor, thapsigargin nearly abolished nortriptyline-induced Ca2+ release. Conversely, pretreatment with nortriptyline greatly reduced the inhibitor-induced [Ca2+]i rise, suggesting that nortriptyline released Ca2+ from the endoplasmic reticulum. Inhibition of phospholipase C did not change the nortriptyline-induced [Ca2+]i rise. Nortriptyline at a concentration of 10 µM increased viability in a Ca2+-independent manner. At 50 µM, nortriptyline killed 45% of cells. Nortriptyline at 10 µM did not induce apoptosis, but at 50 µM induced significant apoptosis measured by propidium iodide staining. Together, in PC3 cells, nortriptyline induced [Ca2+]i rises by causing the phospholipase C-independent Ca2+ release from the endoplasmic reticulum and Ca2+ influx via the protein kinase C-sensitive pathway. Nortriptyline also induced both cell proliferation and death in a concentration-dependent manner. Apoptosis was involved in the cell death. Drug Dev Res 71:323–330, 2010. © 2010 Wiley-Liss, Inc.