We have previously reported that 15-hydroxyeicosatetraenoic acid (15-HETE), a metabolite of arachidonic acid by 15-lipoxygenase, causes pulmonary vasoconstriction via increasing the intracellular Ca2+ concentration ([Ca2+]i). However, the multiple sources of Ca2+ that contribute to Ca2+ elevation during and after 15-HETE exposure have not been investigated. In the present study, pulmonary arterial ring technique and confocal laser scanning microscope were used to investigate the origin of Ca2+. 15-HETE (1 μM) elicited an increase in [Ca2+]i in pulmonary artery smooth muscle cells in a time-dependent manner under both normal and hypoxic condition. The increases were composed of an initial rapid rise followed by a slow increase in the present of extracellular Ca2+. The initial rapid phase was attenuated by inositol 1,4,5-triphosphate (IP3) receptor antagonist 2-aminoethoxydiphenyl borate (2-APB) and ryanodine receptor-operated Ca2+ store depletion agent caffeine; the slow increasing phase and the constriction of pulmonary arterial ring were significantly inhibited by voltage-operated Ca2+ channel blocker nifedipine or transient receptor potential canonical (TRPC) channel blocker La3+, and almost completely diminished in Ca2+-free external solution, suggesting that the initial phase depends on intracellular Ca2+ store and the second phase relies on extracellular Ca2+. Interestingly, the effect of caffeine and La3+ but not nifedipine were diminished in the present of 2-APB. Thus, these results suggest that 15-HETE mobilizes Ca2+ signaling through: 1) Ca2+ release immediately from Ca2+ stores via activation of IP3 receptor and, subsequently that of ryanodine receptor, 2) the depletion of Ca2+ through CCE leading to the activation of TRPC, and 3) Ca2+ entry through L-type Ca2+ channels.