Precapillary arteries constrict during alveolar hypoxia in a response known as hypoxic pulmonary vasoconstriction (HPV). The mechanism by which pulmonary arterial smooth muscle cells (PASMCs) detect a decrease in Po(2) and trigger contraction is not fully understood. Previous studies in cultured PASMCs show that hypoxia induces an increase in reactive oxygen species (ROS) production, but these results may not reflect responses of PASMCs in their native tissue environment. We therefore assessed hypoxia-induced changes in cytosolic ROS in PASMCs of precision-cut mouse lung slices expressing the redox-sensitive protein, RoGFP. Superfusion of lung slices with hypoxic media (1.5% O(2)) resulted in a significant oxidation of RoGFP from normoxic baseline that was attenuated by overexpression of cytosolic catalase. Hypoxic superfusion also increased [Ca(2+)](i) above normoxic baseline; this response was significantly attenuated by cytosolic catalase overexpression or by the administration of EUK134, a synthetic SOD-catalase mimetic. The hypoxia-induced increase in [Ca(2+)](i) was abolished in the absence of extracellular Ca(2+), indicating that ROS signals trigger entry of extracellular calcium. Collectively, these results indicate that an increase in cytosolic ROS signaling is required for the increase in [Ca(2+)](i) in PASMCs in precision-cut mouse lung slices during the acute HPV response.