We have previously reported that hypoxia attenuates cGMP-dependent protein kinase (PKG) mediated relaxation in pulmonary vessels (Am J Physiol 279:L611-8, 2003). To determine if hypoxia-induced reactive oxygen and nitrogen species (ROS/RNS) may be involved in the downregulation of PKG-mediated relaxation, ovine fetal intrapulmonary veins were exposed to 4h of normoxia or hypoxia, with or without scavengers of ROS (NAC) or peroxynitrite (quercetin and Trolox) and preconstricted with endothelin-1. Hypoxia decreased relaxation response to 8-Br-cGMP, PKG protein expression and kinase activity while increasing tyrosine nitration in PKG. However, ROS/RNS scavengers prevented these changes. To determine if increased PKG nitration itself results in diminished PKG activity, pulmonary vein smooth muscle cells (PVSMC) were exposed to shorter-term hypoxia (30min), which led to increased PKG nitration and decreased PKG activity without altering PKG protein expression. Increased DCFH-DA fluorescence in PVSMC after 4h or 30min of hypoxia was not observed in the presence of NAC, quercetin or Trolox, suggesting increased ROS/RNS production. Increased PKG nitration and the associated decrease in PKG activity in PVSMC after 30min of hypoxia were also reversed upon re-oxygenation. The consequences of PKG nitration were assessed by exposing purified PKG1 to peroxynitrite, which caused increased 3-nitrotyrosine immunoreactivity and inhibition of kinase activity. Our data suggest that after short-term exposure to hypoxia (30min), reversible covalent modification of PKG by hypoxia-induced reactive species may be an important mechanism by which relaxation response to cGMP is regulated. However, after longer-term exposure to hypoxia (4h), both PKG nitration and decreased PKG expression are involved.