Reactive oxygen species (ROS) generated from NADPH oxidases and mitochondria have been implicated as key messengers for pulmonary vasoconstriction and vascular remodeling induced by agonists and hypoxia. Since Ca²⁺ mobilization is essential for vasoconstriction and cell proliferation, we sought to characterize the Ca²⁺ response and to delineate the Ca²⁺ pathways activated by hydrogen peroxide (H₂O₂) in rat intralobar pulmonary arterial smooth muscle cells (PASMCs). Exogenous application of 10 µM to 1 mM H₂O₂ elicited concentration-dependent increase in [Ca²⁺]_i in PASMCs, with an initial rise followed by a plateau or slow secondary increase. The initial phase was related to intracellular release. It was attenuated by the inositol trisphosphate (IP₃) receptor antagonist 2-aminoethyl diphenylborate, ryanodine or thapsigargin, but was unaffected by the removal of Ca²⁺ in external solution. The secondary phase was dependent on extracellular Ca²⁺ influx. It was unaffected by the voltage-gated Ca²⁺ channel blocker nifedipine or the non-selective cation channel blockers SK&F 96365 and La³⁺; but inhibited concentration-dependently by millimolar Ni²⁺; and potentiated by the Na⁺-Ca²⁺ exchange inhibitor KB-R7943. H₂O₂ did not alter the rate of Mn²⁺ quenching of fura-2, suggesting store- and receptor-operated Ca²⁺ channels were not involved. By contrast, H₂O₂ elicited a sustained inward current carried by Na⁺ at -70 mV, and the H₂O₂-induced current was inhibited by Ni²⁺. These results suggest that H₂O₂ mobilizes intracellular Ca²⁺ through multiple pathways, including IP₃- and ryanodine receptor-gated Ca²⁺ stores, and Ni²⁺-sensitive cation channels. Activation of these Ca²⁺ pathways may play important roles in ROS signaling in PASMCs.