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 Ca(2+) mobilization is essential for vasoconstriction and cell proliferation, we sought to characterize the Ca(2+) response and to delineate the Ca(2+) pathways activated by hydrogen peroxide (H(2)O(2)) in rat intralobar pulmonary arterial smooth muscle cells (PASMCs). Exogenous application of 10 microM to 1 mM H(2)O(2) elicited concentration-dependent increase in intracellular Ca(2+) concentration 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 (IP(3)) receptor antagonist 2-aminoethyl diphenylborate, ryanodine, or thapsigargin, but was unaffected by the removal of Ca(2+) in external solution. The secondary phase was dependent on extracellular Ca(2+) influx. It was unaffected by the voltage-gated Ca(2+) channel blocker nifedipine or the nonselective cation channel blockers SKF-96365 and La(3+), but inhibited concentration dependently by millimolar Ni(2+), and potentiated by the Na(+)/Ca(2+) exchange inhibitor KB-R 7943. H(2)O(2) did not alter the rate of Mn(2+) quenching of fura 2, suggesting store- and receptor-operated Ca(2+) channels were not involved. By contrast, H(2)O(2) elicited a sustained inward current carried by Na(+) at -70 mV, and the current was inhibited by Ni(2+). These results suggest that H(2)O(2) mobilizes intracellular Ca(2+) through multiple pathways, including the IP(3)- and ryanodine receptor-gated Ca(2+) stores, and Ni(2+)-sensitive cation channels. Activation of these Ca(2+) pathways may play important roles in ROS signaling in PASMCs.