Rho kinase (ROCK)-dependent vasoconstriction is implicated as a major contributing factor to chronic hypoxia (CH)-induced pulmonary hypertension. This component of pulmonary hypertension is associated with arterial myogenicity and increased vasoreactivity to both receptor-mediated agonists and depolarizing stimuli resulting from ROCK-dependent myofilament Ca²⁺ sensitization. Based on separate lines of evidence that CH increases pulmonary arterial superoxide (O₂^-) generation and that O₂^- stimulates RhoA/ROCK signaling in vascular smooth muscle (VSM), we hypothesized that depolarization-induced O₂^- generation mediates enhanced RhoA-dependent Ca²⁺ sensitization in pulmonary VSM following CH. To test this hypothesis, we determined effects of the ROCK inhibitor, HA-1077, and the O₂^- specific spin trap, tiron, on vasoconstrictor reactivity to depolarizing concentrations of KCl in both isolated lungs and Ca²⁺-permeablized, pressurized small pulmonary arteries from control and CH (4 wk at 0.5 atm.) rats. Using the same vessel preparation, we examined effects of CH on KCl-dependent VSM membrane depolarization using sharp electrodes and O₂^- generation with the fluorescent indicator dihydroethidium. Finally, we investigated the contribution of O₂^- to depolarization-induced RhoA activation using a RhoA-GTP pull-down assay. We found that CH augmented KCl-dependent vasoconstriction through a Ca²⁺ sensitization mechanism that was inhibited by both HA-1077 and tiron. Furthermore, CH caused VSM membrane depolarization that persisted with increasing concentrations of KCl, enhanced KCl-induced O₂^- generation and augmented depolarization-dependent RhoA activation in an O₂^--dependent manner. These findings reveal a novel mechanistic link between VSM membrane depolarization, O₂^- generation and RhoA activation that mediates enhanced myofilament Ca²⁺ sensitization and pulmonary vasoconstriction following CH.