Phase I of the hypoxic pulmonary vasoconstriction (HPV) response begins upon transition to hypoxia and involves an increase in cytosolic calcium ([Ca²⁺]_i). Phase II develops during prolonged hypoxia and involves increases in constriction without further increases in [Ca²⁺]_i, suggesting an increase in Ca²⁺ sensitivity. Prolonged hypoxia activates RhoA and RhoA kinase, which may increase Ca²⁺ sensitivity, but the mechanism is unknown. We previously found that reactive oxygen species (ROS) trigger Phase I. We therefore asked whether ROS generation during prolonged hypoxia activates RhoA in PA smooth muscle cells (PASMCs) and endothelial cells (PAECs) during Phase II. By using a cytosolic redox sensor, RoGFP, we detected increased oxidant signaling in prolonged hypoxia in PASMCs (29.8 ± 1.3% to 39.8 ± 1.4%) and PAECs (25.9 ± 2.1% to 43.7.9 ± 3.5%), which was reversed on the return to normoxia and was attenuated with EUK-134 in both cell types. RhoA activity increased in PASMCs and PAECs during prolonged hypoxia (6.4 ± 1.2-fold and 5.8 ± 1.6-fold) and with exogenous H₂O₂ (4.1- and 2.3-fold, respectively). However, abrogation of the ROS signal in PASMCs or PAECs with EUK-134 or anoxia failed to attenuate the increased RhoA activity. Thus, the ROS signal is sustained during prolonged hypoxia in PASMCs and PAECs, and this is sufficient but not required for RhoA activation. Antioxid. Redox Signal. 12, 603–610.