Ca2+ signaling in vascular smooth muscle cells involves dynamic interactions between multiple Ca2+ influx and release pathways. Ca2+ release pathways include the IP3 receptor-, ryanodine receptor-, and nicotinic acid adenine dinucleotide phosphate (NAADP) receptor-gated Ca2+ stores. Recent studies demonstrated that the two-pore channels (TPC1 and TPC2) are NAADP receptors located in the endo-/lysosomes. Here we quantified the expression of TPC channels and characterized the NAADP-mediated Ca2+ signals in vascular tissues. Western blot analysis and real-time RT-PCR detected TPC1 and TPC2 protein and mRNA respectively in aorta, pulmonary, mesenteric, renal, femoral, tail, and cerebral artery of rat, with TPC1 mRNA level five to ten-fold higher than that of TPC2. Application of the membrane permeable NAADP-AM to pulmonary arterial smooth muscle cells (PASMCs) elicited concentration-dependent increase in global [Ca2+]i. The Ca2+ response was independent of extracellular Ca2+ influx, and was unaffected by the IP3 receptor blocker xestospongin C. In contrast, the NAADP-induced Ca2+ response was partially inhibited by ryanodine or thapsigargin, and was abolished by the NAADP antagonist NED-19 or the acidic vacuolar H+-ATPase bafilomycin A. Moreover, NAADP caused dramatic increase in local Ca2+ release events, which was inhibited by ryanodine or NED-19. The temporal and spatial properties of the NAADP-induced local release events were comparable to the spontaneous Ca2+ sparks. Similar observations were also made in aortic smooth muscle cells. Our results, hence, show that the NAADP channels, TPC1 and TPC2, are functionally expressed in vascular smooth muscle; and Ca2+ signals from the NAADP channel-gated lysosomal stores cross-activate ryanodine receptors to amplify Ca2+ release in pulmonary arterial and aortic smooth muscle cells.