Neuropeptide Y (NPY), a neurochemical found in high concentrations within hypothalamic neurons, is believed to participate in the control of eating behavior and body energy balance and elicits a powerful eating response when injected into the hypothalamus. To delineate precisely the locus of this effect, NPY (78 pmol) or its artificial cerebrospinal fluid vehicle was injected in the extremely small volume of 10 nl through chronic guide cannulae into an array of 47 different hypothalamic areas in satiated rats and the elicited food intake was measured. To determine the anatomical resolution of this technique, the spread and recovery of [125I]NPY injected in 10 nl was also assessed. Results indicate that as much as 95% of the injected label was recovered within the brain tissue and guide cannulae and that 100% of the tissue label was localized to within 0.8 mm of the injection site. Behavioral results show that the perifornical hypothalamus (PFH), at the level of the caudal paraventricular nucleus, is the most sensitive hypothalamic site for NPY-induced eating. NPY there elicited mean increases in food intake of 12.5 g over baseline at 1 h and 20.0 g at 4 h postinjection. Injections bracketing the PFH in all directions were substantially less effective. Additionally, significant effects were also observed in at least seven other sites that were distributed throughout the hypothalamus. These findings suggest both that the PFH may be the primary hypothalamic site containing feeding-related NPY-sensitive receptors and that other sites distributed within the hypothalamus also can mediate NPY's effects.