TRPM2 is a calcium-permeable nonselective cation channel that is opened by the binding of ADP-ribose (ADPR) to a C-terminal nudix domain. Channel activity is further regulated by several cytosolic factors, including cyclic ADPR (cADPR), nicotinamide adenine dinucleotide phosphate (NAADP), Ca 2+ and calmodulin (CaM), and adenosine monophosphate (AMP). In addition, intracellular ions typically used in patch-clamp experiments such as Cs + or Na + can alter ADPR sensitivity and voltage dependence, complicating the evaluation of the roles of the various modulators in a physiological context. We investigated the roles of extra- and intracellular Ca 2+ as well as CaM as modulators of ADPR-induced TRPM2 currents under more physiological conditions, using K + -based internal saline in patch-clamp experiments performed on human TRPM2 expressed in HEK293 cells. Our results show that in the absence of Ca 2+ , both internally and externally, ADPR alone cannot induce cation currents. In the absence of extracellular Ca 2+ , a minimum of 30 nM internal Ca 2+ is required to cause partial TRPM2 activation with ADPR. However, 200 μM external Ca 2+ is as efficient as 1 mM Ca 2+ in TRPM2 activation, indicating an external Ca 2+ binding site important for proper channel function. Ca 2+ facilitates ADPR gating with a half-maximal effective concentration of 50 nM and this is independent of extracellular Ca 2+ . Furthermore, TRPM2 currents inactivate if intracellular Ca 2+ levels fall below 100 nM irrespective of extracellular Ca 2+ . The facilitatory effect of intracellular Ca 2+ is not mimicked by Mg 2+ , Ba 2+ , or Zn 2+ . Only Sr 2+ facilitates TRPM2 as effectively as Ca 2+ , but this is due to Sr 2+ -induced Ca 2+ release from internal stores rather than a direct effect of Sr 2+ itself. Together, these data demonstrate that cytosolic Ca 2+ regulates TRPM2 channel activation. Its facilitatory action likely occurs via CaM, since the addition of 100 μM CaM to the patch pipette significantly enhances ADPR-induced TRPM2 currents at fixed [Ca 2+ ] i and this can be counteracted by calmidazolium. We conclude that ADPR is responsible for TRPM2 gating and Ca 2+ facilitates activation via calmodulin.