This study determined whether nociceptin/orphanin FQ (NOC/oFQ) generates superoxide anion (O₂) in a protein kinase C (PKC)-dependent manner and whether such production contributes to hypoxic-ischemic (H-I) impairment of N-methyl-D-aspartate (NMDA)-induced pial artery dilation in newborn pigs equipped with closed cranial windows. Superoxide dismutase (SOD)-inhibitable nitroblue tetrazolium (NBT) reduction was an index of O₂ generation. Under non-H-I conditions, topical NOC/oFQ (10¹⁰ M, concentration present in cerebrospinal fluid after I or H-I) increased SOD-inhibitable NBT reduction from 1 ± 1 to 20 ± 3 pmol/mm². PKC inhibitors staurosporine and chelerythrine (10⁷ M) blunted NBT reduction (1 ± 1 to 7 ± 2 pmol/mm² for chelerythrine), whereas the NOC/oFQ receptor antagonist [F/G]NOC/oFQ (1-13)-NH₂ (10⁶ M) blocked NBT reduction. [F/G]NOC/oFQ(1-13)-NH₂ and staurosporine also blunted the NBT reduction observed after I or H-I. NMDA (10⁸, 10⁶ M)-induced pial artery dilation was reversed to vasoconstriction after H-I. The NOC/oFQ antagonist staurosporine and free radical scavengers partially prevented this impaired dilation (sham: 9 ± 1 and 16 ± 1; H-I: 5 and 10 ± 1; H-I staurosporine pretreated: 3 ± 1 and 6 ± 1%). These data show that NOC/oFQ increased O₂ production in a PKC-dependent manner and contributed to this production after insult and that NOC/oFQ contributed to impaired NMDA-induced pial artery dilation after H-I, suggesting, therefore, that PKC-dependent O₂ generation by NOC/oFQ links NOC/oFQ release to impaired NMDA dilation after H-I.