Phrenic long-term facilitation (pLTF) following acute intermittent hypoxia (AIH) is a form of spinal, serotonin-dependent synaptic plasticity that requires reactive oxygen species (ROS) formation. We tested the hypothesis that spinal NADPH oxidase activity is a necessary source of ROS for pLTF. Sixty minutes post-AIH (three 5-min episodes of 11% O(2), 5 min intervals), integrated phrenic and hypoglossal (XII) nerve burst amplitudes were increased from baseline, indicative of phrenic and XII LTF. Intrathecal injections (approximately C(4)) of apocynin or diphenyleneiodonium chloride (DPI), two structurally and functionally distinct inhibitors of the NADPH oxidase complex, attenuated phrenic, but not XII, LTF. Immunoblots from soluble (cytosolic) and particulate (membrane) fractions of ventral C(4) spinal segments revealed predominantly membrane localization of the NADPH oxidase catalytic subunit, gp91(phox), whereas membrane and cytosolic expression were both observed for the regulatory subunits, p47(phox) and RAC1. Immunohistochemical analysis of fixed tissues revealed these same subunits in presumptive phrenic motoneurons of the C(4) ventral horn, but not in neighbouring astrocytes or microglia. Collectively, these data demonstrate that NADPH oxidase subunits localized within presumptive phrenic motoneurons are a major source of ROS necessary for AIH-induced pLTF. Thus, NADPH oxidase activity is a key regulator of spinal synaptic plasticity, and may be a useful pharmaceutical target in developing therapeutic strategies for respiratory insufficiency in patients with, for example, cervical spinal injury.