Recent studies have revealed the importance of two-pore segment channels (TPCs) in mediating NAADP-evoked Ca2+ release from acidic organelles. The Ca2+ signal initiated from the acidic stores can recruit additional Ca2+ release from endoplasmic reticulum (ER) via Ca2+-induced Ca2+ release (CICR). Both humans and mice express two TPCs, TPC1 and TPC2 whereas many other vertebrates express also TPC3. We have shown that each TPC subtype may be targeted to different acidic organelles, with TPC1 predominantly localized to a subpopulation of endosomes while TPC2 almost exclusively expressed in lysosomes. Consistent with these expression patterns and the fact that endosomes are smaller and less clustered than lysosomes, TPC1 mediated Ca2+ release in response to 10 nM NAADP in a manner that was spatially restricted, insensitive to thapsigargin, and unable to trigger global Ca2+ transient. By contrast, Overexpression of TPC2 resulted in a biphasic Ca2+ response to NAADP with a pacemaking phase followed by a large secondary and global Ca2+ transient and the latter response was largely attenuated by ER store depletion with thapsigargin. Therefore, only TPC2 appears to couple to the ER by CICR. Interestingly, the expression of chicken and rabbit TPC3 in HEK293 cells yielded distinct subcellular localizations and functional data are consistent with differential organelle targeting. Our data suggest that cross-talk between acidic stores and ER is governed by the capacity of local Ca2+ signals from acidic Ca2+ stores to summate, and in a manner that may determine whether the threshold for CICR from ER is breached.