Abstract: The effect of the endogenous cannabinoid anandamide on cytosolic free Ca2+ concentration ([Ca2+]i) and proliferation is largely unknown. This study examined whether anandamide altered Ca2+ levels and caused Ca2+-dependent cell death in Madin-Darby canine kidney (MDCK) cells. [Ca2+]i and cell death were measured using the fluorescent dyes fura-2 and WST-1 respectively. Anandamide at concentrations above 5 μM increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signal was reduced by 78% by removing extracellular Ca2+. The anandamide-induced Ca2+ influx was insensitive to L-type Ca2+ channel blockers and the cannabinoid receptor antagonist AM 251, but was inhibited differently by aristolochic acid, WIN 55,212-2 (a cannabinoid receptor agonist), phorbol ester, GF 109203X and forskolin. After pretreatment with thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor), anandamide-induced Ca2+ release was inhibited. Inhibition of phospholipase C with U73122 did not change anandamide-induced Ca2+ release. At concentrations of 100 μM and 200 μM, anandamide killed 50% and 95% cells, respectively. The cytotoxic effect of 100 μM anandamide was completely reversed by pre-chelating cytosolic Ca2+ with BAPTA. Collectively, in MDCK cells, anandamide induced [Ca2+]i rises by causing Ca2+ release from endoplasmic reticulum and Ca2+ influx from extracellular space. Furthermore, anandamide can cause Ca2+-dependent cytotoxicity in a concentration-dependent manner.