Production of nitric oxide (NO) in the hypothalamic paraventricular nucleus (PVN) was examined by microdialysis in rats subjected to immobilization (IMO) stress. A dialysis probe was implanted in the posterior magnocellular subdivision of the PVN and nitrite (NO₂^-), an oxidized product of NO, was measured continuously. NO₂^- concentration in dialysate was enhanced to 156% after 30-min IMO compared with the NO₂^- level before IMO. Intraperitoneal administration of N^G-monomethyl-L-arginine (L-NMMA, 10 mg/kg), a NO synthase inhibitor, before IMO completely inhibited the increase of NO production that IMO was to induce. Depletion of catecholamines innervating the PVN by an injection of 6-hydroxydopamine into the lateral ventricle before the microdialysis had no suppressive effect on the increase of NO production by IMO. In contrast, NO₂^- levels in the PVN was lowered by continuous perfusion of the solution containing ionotropic glutamate receptor antagonists 2-amino-5-phosphonovaleric acid (AP5, 500 µM) and 6-cyano-7-nitroquinoxaline-2, 3 dione (CNQX, 50 µM) through the dialysis probe, and the IMO-induced increase of NO production was attenuated by the treatment. These results suggest that catecholaminergic drive to the hypothalamus is not necessary for the IMO-induced increase of NO production and that ionotropic glutamate receptors play a role in the basal and IMO-induced NO production.