We have recently demonstrated that chronic hypoxia (CH) attenuates nitric oxide (NO)-mediated decreases in pulmonary vascular smooth muscle (VSM) free calcium concentration ([Ca²⁺]_i) and promotes NO-dependent VSM Ca²⁺-desensitization. The objective of the current study was to identify potential mechanisms by which CH interferes with regulation of [Ca²⁺]_i by NO. We hypothesized that CH impairs NO-mediated inhibition of store-operated (capacitative) Ca²⁺ entry (SOCE) or receptor-operated Ca²⁺ entry (ROCE) in pulmonary VSM. To test this hypothesis, we examined effects of the NO donor, spermine NONOate, on SOCE resulting from depletion of intracellular Ca²⁺ stores with cyclopiazonic acid, and on UTP-induced ROCE in isolated, endothelium-denuded and pressurized pulmonary arteries (213 ± 8 µm inner diameter) from control and CH (4 wk at 0.5 atm) rats. Arteries were loaded with fura-2 AM to continuously monitor VSM [Ca²⁺]_i. We found that the change in [Ca²⁺]_i associated with SOCE and ROCE was significantly reduced in vessels from CH animals. Furthermore, spermine NONOate diminished SOCE and ROCE in vessels from control, but not CH animals. We conclude that NO-mediated inhibition of SOCE and ROCE is impaired following CH-induced pulmonary hypertension.