Neuropeptide Y (NPY) and corticotropin-releasing factor (CRF) are present at high concentrations in the hypothalamus where they mediate important endocrine and autonomic functions. Morphological and physiological studies have suggested an interaction between these peptides, and opposing actions of CRF and NPY have been reported on feeding and other behaviors. This study investigated the effect of CRF on NPY release in vivo, measured by push-pull techniques, in the anesthetized rat. Push-pull probes implanted into the paraventricular nucleus of the hypothalamus (PVN) were perfused with modified Ringer solution containing bovine serum albumin at 15 μl/min, and the perfusate was lyophilized prior to NPY radioimmunoassay. NPY overflow from the rat PVN was increased threefold by perfusion of a depolarizing concentration of potassium (50 mmol/L KCl). When CRF was administered into the PVN via the pushpull cannula at 1 or 5 μg/ml, dose-dependent increases in NPY overflow of two- and fivefold were observed (p < 0.05). These increases were abolished by prior intracerebroventricular (i.c.v.) administration of the CRF antagonist [D-Phe12,Nle21,38,CαMeLeu32] CRF (12-41) at 1 or 5 μg/μl, respectively. NPY overflow returned promptly to resting levels following CRF administration. In contrast, when CRF was administered by i.c.v. bolus at a similar total dose (2 μg), no significant effect on NPY overflow was observed. These data provide in vivo evidence for an interaction between CRF and NPY at the level of the PVN.