Nicotinic acid adenine dinucleotide phosphate (NAADP) potently releases Ca2+ from acidic intracellular Ca2+ stores. There is evidence that two pore channels (TPCs), a family of ion-channel localised to acidic stores, may be responsible for NAADP-mediated Ca2+-release. In humans there are two functional isoforms; type-1 (TPC1) and type-2 (TPC2). The physiological roles of these channels are not fully characterised although NAADP has been implicated in a range of processes including fertilization, exocytosis, autophagy and cardiac function. Understanding the biophysical properties and pharmacology of TPCs will enable us to determine the physiological roles that these channels play and to begin to understand the interplay between different TPC isoforms. Here we incorporate purified recombinant human TPC1 and TPC2 proteins into planar phosphatidylethanolamine lipid bilayers under voltage-clamp conditions according to previously described techniques (1). We have then compared the ion-selectivity and regulation of gating of these two channels. Regarding channel activation we show that TPC2 and TPC1 are both ligand-gated ion channels. NAADP is an absolute requirement for TPC2 activation (1), whereas TPC1 can be activated by either NAADP or cytosolic Ca2+ (2). Regarding ion conduction, TPC1 and TPC2 are both non-selective cation channels. TPC2 however is more permeable to Ca2+ over any other ion that we tested (1), whereas the relative permeability of TPC1 under bi-ionic conditions decreases in the order H+>>K+>Na+≥Ca2+ (2). We suggest that TPC2 is the primary NAADP-activated Ca2+-release channel of acidic Ca2+-stores and that TPC1 plays a less significant role in Ca2+-release but may be important in providing proton leak. In mammalian cells it is accepted that NAADP-mediated Ca2+ responses are desensitised by the application of high concentrations of NAADP and that Ned-19 is a selective blocker of cellular NAADP-induced Ca2+ release (3). Using K+ as the permeant ion (210 mM symmetrical KCl solutions), and in the presence of 10 µM cytosolic Ca2+, we show that the addition of 1 μM Ned-19 or 1 mM NAADP inhibits TPC2 function, whereas they have no significant effect on TPC1 gating under these conditions. This suggests that the pharmacological regulation of TPC2 and TPC1 is different. Our data reveal that although TPC1 and TPC2 share certain functional similarities, there are important differences in their ion-selectivity and regulation of gating that will enable the endolysosomal system to respond to physiological needs by integrating a variety of cellular signals.