Cellular redox change regulates pulmonary vascular tone. We examined the effect of thiol oxidation on excitation-contraction coupling in isolated pulmonary artery (PA) rings and potential mechanisms by which thiol oxidation regulates pulmonary vascular tone. The thiol oxidant diamide produced a dose-dependent relaxation in PA rings; the IC₅₀ was 335 and 58 µM for 40 mM K⁺- and 2 µM phenylephrine (PE)-induced PA contraction, respectively. The diamide-mediated pulmonary vasodilation was neither affected by functional removal of endothelium nor by 8-Br-cGMP and HA-1004. Raising extracellular [K⁺] (from 20 to 80 mM) attenuated diamide-induced PA relaxation. Passive store depletion by cyclopiazonic acid (CPA) and active store depletion by PE (in the absence of external Ca²⁺) both induced PA contraction due to capacitative Ca²⁺ entry (CCE). Diamide significantly attenuated CCE-induced PA contraction due to active and passive store depletion. While the active tension induced by passive store depletion was comparable, the active tension induced by active store depletion was 3.5 fold greater in right branches than in left branches. These data indicate that thiol oxidation causes pulmonary vasodilation by activating K⁺ channels and inhibiting store-operated Ca²⁺ channels, which subsequently attenuate Ca²⁺ influx and decrease [Ca²⁺]_cyt in pulmonary artery smooth muscle cells. The mechanisms involved in thiol oxidation-mediated pulmonary vasodilation or activation of K⁺ channels and inhibition of store-operated Ca²⁺ channels appear to be independent of functional endothelium and of the cGMP-PKG pathway.