Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent Ca²⁺ mobilising messenger in mammalian and non-mammalian cells. Studies on a variety of cell types suggest that NAADP evokes Ca²⁺ release from a lysosome-related store and via activation of a receptor distinct from either ryanodine receptors or inositol 1,4,5-trisphosphate receptors (IP₃R; 1,2). Consistent with this view, studies on pulmonary arterial smooth muscle cells (PASMC) have shown that NAADP elicits Ca²⁺ signals by mobilizing lysosome-related Ca²⁺ stores (3). Recent investigations have suggested that the two-pore channel subtype 2 (TPC2) acts as an NAADP receptor in HEK293 cells that stably over-express human TPC2 (hTPC2; 4). We therefore investigated the possible role of TPC2 in NAADP-mediated Ca²⁺ release in PASMC. All experiments were performed in accordance with the United Kingdom Animals (Scientific Procedures) Act 1986. PASMCs were isolated from male Wistar rats (150-300 g) that were humanely killed. Ca²⁺ imaging using Fura-2 fluorescence, and fluorescent imaging and analysis of subcellular labelling was carried out following previously described protocols (3). RT-PCR analysis showed that TPC2 is expressed in PASMCs. Furthermore, in HEK293 cells that stably over-express hTPC2 and in acutely isolated rat PASMC deconvolution microscopy demonstrated that fluorescent labelling of TPC2 colocalised with LysoTracker Red labelled lysosomes (n = 3). HEK293 cells over-expressing hTPC2 exhibited a marked increase in the Fura-2 fluorescence ratio (F₃₄₀/F₃₈₀) from 0.75 ± 0.03 to 1.67 ± 0.15 (mean ± S.E.M., n = 44) in response to intracellular dialysis of NAADP (10 nM) from a patch pipette. Similarly, under control conditions, 10 nM NAADP elicited a global Ca²⁺ wave in PASMC increasing the F₃₄₀/F₃₈₀ ratio from 0.64 ± 0.04 to 1.81 ± 0.13 (n = 22). Consistent with the notion that NAADP mobilises Ca²⁺ from a lysosome-related acidic store, the response to 10 nM NAADP was abolished by disruption of the lysosomal proton gradient using bafilomycin A1 (100 nM) in both PASMC (n = 7) and hTPC2 over-expressing HEK293 cells (n = 11). Furthermore, a specific NAADP antagonist, Ned-14 (100 μM) abolished NAADP-mediated Ca²⁺ release in both hTPC2 over-expressing HEK293 cells (n = 5) and PASMCs (n = 4). These data demonstrate that TPC2 exhibits analogous functional characteristics in both PASMCs and hTPC2 over-expressing HEK293 cells. These findings provide further support for the view that TPC2 is the molecular target of NAADP in both cell types.