We have previously shown that cyclopiazonic acid, a sarco / endoplasmic reticulum Ca2+ ATPase (SERCA) antagonist, blocks isoprenaline-induced dilation of pulmonary arterial smooth muscle that is mediated by Ca2+ release from the sarcoplasmic reticulum (SR) via ryanodine receptors (RyRs)(1), but fails to block vasoconstriction induced by SR Ca2+ release via RyRs(2). We investigated the possibility that targeting of SERCA and RyR to discrete SR regions may confer functionally segregated SR compartments within pulmonary arterial smooth muscle cells; procedures accorded with current UK legislation. Immunofluorescence labelling (for methods(3)) of isolated pulmonary arterial smooth muscle cells revealed striking differences in the spatial distribution of SERCA and RyR. SERCA2a was mostly restricted to a region within 1.5 µm of the nucleus (perinuclear region, 0.125 ± 0.019 µm3 per µm3; extraperinuclear region, 0.003 ± 0.001 µm3 per µm3; subplasmalemmal region, 0.003 ± 0.002 µm3 per µm3) as was RyR3 labelling (perinuclear region, 0.1 ± 0.01 µm3 per µm3; extraperinuclear region, 0.02 ± 0.01 µm3 per µm3; subplasmalemmal region, 0.003 ± 0.001 µm3 per µm3). By contrast, SERCA2b labelling was primarily found within 1.5 µm of the plasma membrane (perinuclear region, 0.033 ± 0.011µm3 per µm3; extraperinuclear region, 0.010 ± 0.003µm3 per µm3; subplasmalemmal region, 0.106 ± 0.016µm3 per µm3) where labelling for RyR1 was maximal (perinucleaar region, 0.0211 ± 0.005 µm3 per µm3; extraperinuclear region, 0.036 ± 0.007 µm3 per µm3; subplasmalemmal region, 0.046 ± 0.008 µm3 per µm3). The majority of labelling for RyR2 lay in between these two regions of the cell (perinuclear region, 0.022 ± 0.01 µm3 per µm3; extraperinuclear region, 0.072 ± 0.01 µm3 per µm3; subplasmalemmal region, 0.045 ± 0.01 µm3 per µm3). Application of the vasoconstrictor endothelin-1 (100 nM) induced global Ca2+ waves in pulmonary arterial smooth muscle cells (reported by the Fura-2 fluorescence ratio (F340 / F380) which increased from 0.49 ± 0.02 to 1.83 ± 0.03 (n = 31)). Responses were markedly attenuated upon depletion of SR Ca2+ stores by pre-incubation (20 min) of cells with the SERCA antagonist thapsigargin (1 μM; F340 / F380 increasing from 0.52 ± 0.02 to 0.56 ± 0.03 (n = 15, P ≤ 0.01)), but remained unaffected by pre-incubation with cyclopiazonic acid (10 μM; F340 / F380 increasing from 0.59 ± 0.03 to 1.79 ± 0.05 (n = 31)). We conclude that functionally segregated SR Ca2+ stores exist within pulmonary arterial smooth muscle cells. One sits proximal to the plasma membrane, receives Ca2+ via SERCA2b and likely releases Ca2+ via RyR1 to mediate vasodilation. The other is located centrally, receives Ca2+ via SERCA2a and likely releases Ca2+ via RyR3 and RyR2 to initiate vasoconstriction.