The effect of N-(4-hydroxyphenyl)-arachidonamide (AM-404), a drug commonly used to inhibit the anandamide transporter, on intracellular free Ca 2 + levels ([Ca 2 + ] i ) was studied in Madin Darby canine kidney (MDCK) cells. [Ca 2 + ] i was measured using fura-2 as a Ca 2 + indicator. Between 2 and 40 μM, AM-404 increased [Ca 2 + ] i in a concentration-dependent fashion with an EC 5 0 value of 20 μM. Removal of extracellular Ca 2 + abolished the [Ca 2 + ] i signals. The [Ca 2 + ] i increase was nearly abrogated by 10 μM La 3 + , but was insensitive to 50 μM Ni 2 + and 10 μM of nifedipine, nimodipine, nicardipine, and verapamil. At a concentration that did not increase [Ca 2 + ] i , AM-404 (1 μM) did not alter the [Ca 2 + ] i increases induced by 10 μM ATP and 1 μM bradykinin. AM-404 (5 μM) also increased [Ca 2 + ] i in Chang liver cells, PC3 human prostate cancer cells, BFTC human bladder cancer cells, and MG63 human osteoblast-like cells. Together, this study shows for the first time that AM-404 at concentrations commonly used to inhibit the anandamide transporter in various systems induced an increase in [Ca 2 + ] i in different cell types. The [Ca 2 + ] i increase was solely due to extracellular Ca 2 + influx. Thus caution must be exercised in using AM-404 as a selective inhibitor of the anandamide transporter.