Neuropeptide (NPY) increases feeding when injected into the brain. In this study, we tested the hypothesis that its action might be related to feeding regulation of the orexin and leptin systems in rats. Intracerebroventricular administration of NPY (1 nmol/5 microL) stimulated feeding in rats. Injection of an antibody to orexin-A inhibited feeding, suggesting that endogenous orexin exerts a stimulatory tone on feeding. Intracerebroventricular injection of orexin antiserum before injection of NPY significantly attenuated the feeding response to NPY. On the other hand, ip pretreatment with leptin (2 mg/kg) significantly decreased food intake and inhibited NPY-induced feeding. We then examined whether orexin-containing neurons are activated under the stimulation of feeding in response to intracerebroventricular NPY or suppression of feeding in response to ip leptin, using Fos-like immunoreactivity (FLI) as a marker of neural activation. We observed that FLI was induced in the paraventricular, supraoptic, and dorsomedial nuclei as well as the lateral hypothalamic area (LHA) following administration of NPY. Double staining with anti-Fos and antiorexin antibodies revealed that 23.4% of the orexin-containing neurons in the LHA expressed FLI after NPY injection. Approximately 7.8% of the orexin-positive neurons in the LHA coexpressed Fos after leptin plus NPY. Our data indicate that a functional interaction among NPY, orexin, and leptin exists that may contribute to the central regulation of appetite.