The present study is a test of the hypothesis that endogenous glutamatergic input to the hypothalamic paraventricular nucleus (PVN) is involved in stress-induced activation of the hypothalamic-pituitary-adrenocortical (HPA) axis. We examined whether corticosterone (CORT) responses to restraint stress could be attenuated by bilateral 50 nl microinjections of kynurenic acid (KYN, ionotropic glutamate receptor antagonist) into the medial PVN of conscious rats. Immediately following microinjection, rats were subjected to 30 min restraint, and stress-induced plasma CORT was measured at 30, 60, 120, and 180 min following the onset of restraint. KYN (50 pmol) significantly reduced cumulative CORT responses to acute restraint stress by 24%. In contrast, microinjections centered dorsal to the PVN increased CORT responses by 31%, suggestive of a disinhibition of local PVN-inhibitory input. KYN injections immediately anterior, ventral, or posterior to the PVN had no effect, suggesting an absence of potential diffusion artifacts. These results provide evidence that endogenous glutamate in the PVN and surrounding peri-PVN region plays a physiologically significant role in both generating and limiting glucocorticoid stress responses in awake rats.