Intermittent hypoxia (IH) associated with sleep apneas leads to cardio-respiratory abnormalities which may involve altered neuropeptide signaling. The effects of IH on neuropeptide synthesis have not yet been investigated. Peptidylglycine -amidating monooxygenase (PAM; EC 1.14.17.3) catalyzes the -amidation of neuropeptides which confers biological activity to a large number of neuropeptides. PAM comprises of oxygen-sensitive peptidylglycine -hydroxylating monooxygenase (PHM) and peptidyl--hydroxyglycine -amidating lyase (PAL) activities. Here, we examined whether IH alters neuropeptide synthesis via affecting PAM activity and, if so, by what mechanisms. Experiments were performed on adult male rats exposed to either IH (5% O₂ for 15 sec followed by 21% O₂ for 5 min; 8h/day for up to 10 days)or to continuous hypoxia (CH; 0.4 atm for 10 days). Analysis of the brainstem extracts showed that IH but not CH increased PHM but not PAL activity of PAM and that the increase in PHM activity was associated with a concomitant elevation in the levels of -amidated forms of substance P (SP) and neuropeptide Y (NPY). IH increased the relative abundance of 42 and 35-kDa forms of PHM (~1.6- and 2.7-fold, respectively) suggesting enhanced proteolytic processing of PHM, which appears to be mediated by IH-induced increase in endoprotease activity. Kinetic analysis showed that IH increases the V_max and ascorbate-dependent PHM activity but has no effect on K_m. IH increased the generation of reactive oxygen species (ROS) in the brainstem and systemic administration of anti-oxidant prevented IH-evoked increases in PHM activity, proteolytic processing of PHM, endoprotease activity and elevations in the levels of SP and NPY amides. Taken together, these results demonstrate that IH activates PHM in the rat brainstem via ROS-dependent post-translational proteolytic processing and further suggest that PAM activation may contribute to IH-mediated peptidergic neurotransmission in the rat brainstem.