Stromal interaction molecule 1 (STIM1) is a recently discovered membrane-spanning protein thought to sense lumenal Ca²⁺ in sarco(endo)plasmic reticulum (SR/ER) and transduce activation of Ca²⁺-permeable store-operated channels (SOC) in plasmalemma in response to SR/ER Ca²⁺ depletion. To evaluate the role of STIM1 and a closely related protein, STIM2, in Ca²⁺ signaling of rat distal pulmonary arterial smooth muscle cells (PASMC) during hypoxia, we used fluorescent microscopy and the Ca²⁺-sensitive dye, Fura-2, to measure basal intracellular Ca²⁺ concentration ([Ca²⁺]_i), store-operated Ca²⁺ entry (SOCE), and increases in [Ca²⁺]_i caused by acute hypoxia (4% O₂) or depolarization (60 mmol/L KCl) in cells treated with small interfering RNA targeted to STIM1 (siSTIM1) or STIM2 (siSTIM2). As determined by real-time quantitative polymerase chain reaction analysis (qPCR), STIM1 mRNA was 200-fold more abundant than STIM2 in untreated control PASMC. siSTIM1 and siSTIM2 caused specific and significant knockdown of both mRNA measured by qPCR and protein measured by Western blotting. siSTIM1 markedly inhibited SOCE and the sustained [Ca²⁺]_i response to hypoxia, but did not alter the initial transient [Ca²⁺]_i response to hypoxia, the [Ca²⁺]_i response to depolarization, or basal [Ca²⁺]_i. The only effect of siSTIM2 was a smaller inhibition of SOCE. We conclude that STIM1 was quantitatively more important than STIM2 in activation of SOC in rat distal PASMC, and that the increase in [Ca²⁺]_i induced by acute hypoxia in these cells required SR Ca²⁺ release and STIM1-dependent activation of SOC.