The mechanism by which nicotinic acid adenine dinucleotide phosphate (NAADP) triggers intracellular Ca2+ release has been hotly debated. Two-pore segment channels (TPC1-3, TPCN1-3 for gene name) were recently identified as endolysosome-targeted ion channels (1- 3) that support NAADP-evoked Ca2+ signals from acidic stores. Although TPC3 is expressed by other mammals, only TPC1 and TPC2 are expressed by humans, rats and mice (1, 3). The relative capacity of TPC1 and TPC2 to support coupling, by calcium-induced calcium release (CICR), between acidic stores and the sarco/endoplasmic reticulum has yet to be assessed in detail outside recombinant systems. We sought to address this question using methods described previously (1) and in doing so took advantage of our most recent observation (4), that when stably expressed in HEK293 cells, human (h)TPC1 is preferentially targeted to endosomes, hTPC2 to lysosomes, and rabbit (r)TPC3 to both endosomes and lysosomes. When applied by intracellular dialysis from a patch-pipette (voltage-clamp mode, -40 mV holding potential), 10 nM NAADP evoked robust, global calcium transients in both acutely isolated rat pulmonary arterial myocytes and in hTPC2-expressing HEK293 cells, the Fura-2 fluorescence ratio (F340/F380) increased from 0.35 ± 0.04 to 1.29 ± 0.08 (n=10) and from 0.31±0.02 to 1.22±0.08 (n=40), respectively. In both cell types, NAADP-evoked calcium transients were markedly attenuated by thapsigargin (1μM), but abolished by bafilomycin (1μM) and nifedipine (10μM). Qualitatively, similar results were obtained in relation to NAADP-evoked calcium signals in HEK293 cells that stably expressed the endolysosome targeted rTPC3 (F340/F380 increased from 0.38 ± 0.04 to 1.59 ± 0.24, n=11). By contrast, NAADP evoked highly localised (spatially restricted) calcium transients in hTPC1-expressing HEK293 cells, which remained unaffected in the presence of thapsigargin (F340/F380 increased from 0.30 ± 0.03 to 0.58 ± 0.06, n=8), but were blocked by bafilomycin (1µM) and nifedipine (10µM). Furthermore, NAADP failed to evoke global calcium signals in pulmonary arterial myocytes isolated from Tpcn2 knockout mice, despite the continued expression of TPC1. We conclude that NAADP induces global calcium waves in pulmonary arterial myoctes via lysosome-SR junctions and in a manner supported by TPC2, but not TPC1.