Introduction: In pulmonary hypertension (PH) the increased pulmonary blood pressure exerts a stretching force on pulmonary arterial wall that leads to contraction of pulmonary arterial smooth muscle cells (PASMC), a mechanism called myogenic tone. PASMC can transduce the mechanical stretch signal into a biological response by increasing the intracellular calcium concentration via membrane stretch-activated channels (SAC). Here, we investigate how the subcellular organization of calcium stores in PASMC (sarcoplasmic reticulum (SR), lysosomes and mitochondria) is important for the calcium response to stretch during PH. Methods: PASMC were freshly isolated from normoxic rats (Nx rats) and rats with a pulmonary hypertension induced by an injection of monocrotaline (MCT rats). Inward currents from SAC were recorded after a negative pressure applied by a patch-clamp pipette and calcium variations were recorded with the fluorescent probe indo-1, simultaneously. Cytosolic calcium was also measured with the fluo-4 probe and mitochondrial calcium with the rhod-2 probe by a confocal microscope after an osmotic shock. A pharmacological approach coupled with different coimmunolabelings of ryanodine receptors (RyR) and SERCA2 pumps was used to investigate which RyR subtype is involved in calcium response to stretch. The cellular distribution of lysosomes, mitochondria and sarcoplasmic reticulum was investigated with live cellular probes. Results: PASMC exhibit different segregated calcium stores: a subplasmalemnal SR store with RyR1 and SERCA2b, associated to mitochondria and a perinuclear SR store with RyR3 and SERCA2a. We showed that a stretch of PASMC induces an inward calcium influx through SAC that is amplified by a calcium release by RyR1. This calcium rise is then buffered by mitochondria. In MCT rats, the subcellular organization of RyR subtypes is modified: RyR3 and SERCA2a are not only expressed in a perinuclear area but also in a subplasmalemnal level. This is correlated with a different organization of mitochondria. In MCT rats, this new organisation leads to a greater amplification by all RyR subtypes and to a higher calcium increase induced by stretch. Furthermore, the calcium response to stretch is enhanced by a mechanism independent on extracellular calcium, involving caveolae. Conclusion: This study highlights how the spatial organization of calcium stores in PASMC is important for calcium signaling to stretch and that a modification of this segregation leads to PH in rats. Such a study is relevant for a better understanding of cellular mechanisms underlying PH.