Previous studies have shown that in acutely dispersed canine pulmonary artery smooth muscle cells (PASMCs), depletion of both functionally independent IP₃- and ryanodine-sensitive Ca²⁺ stores activates capacitative Ca²⁺ entry (CCE). The present study aimed to determine if cell culture modifies intracellular Ca²⁺ stores and alters Ca²⁺ entry pathways caused by store-depletion and hypoxia in canine PASMCs. Intracellular calcium concentration ([Ca²⁺]_i) was measured in fura-2 loaded cells. Mn²⁺ quench of fura-2 signal was performed to study divalent cation entry and the effects of hypoxia were examined under oxygen tension of 15-18 mmHg. In acutely isolated PASMCs, depletion of IP₃-sensitive Ca²⁺ stores with CPA did not affect initial caffeine-induced intracellular Ca²⁺ transients but abolished 5-HT-induced Ca²⁺ transients. In contrast, CPA significantly reduced caffeine- and 5-HT-induced Ca²⁺ transients in cultured PASMCs. In cultured PASMCs, store-depletion or hypoxia caused a transient followed by a sustained rise in [Ca²⁺]_i. The transient rise in [Ca²⁺]_i was partially inhibited by nifedipine whereas the nifedipine-insensitive transient rise in [Ca²⁺]_i was inhibited by KB-R7943, a selective inhibitor of reverse mode Na⁺/Ca²⁺ exchanger (NCX). The nifedipine-insensitive sustained rise in [Ca²⁺]_i was inhibited by SKF96365, Ni²⁺, La³⁺ and Gd³⁺. In addition, store-depletion or hypoxia increased the rate of Mn²⁺ quench of fura-2 fluorescence that was also inhibited by these blockers, exhibiting pharmacological properties characteristic of CCE. We conclude that cell culture of canine PASMCs reorganizes IP₃ and ryanodine receptors into a common intracellular Ca²⁺ compartment and depletion of this store or hypoxia activates voltage-operated Ca²⁺ entry, reverse mode NCX and CCE.