Little is known about the mechanism of action behind the orexigenic activity of cannabinoids. Neuropeptide Y (NPY) is one of the most potent orexigenic factors and is a key mediator in the hypothalamic control of food intake. We examined the effect of cannabinoids on NPY release using a rat hypothalamic explant model. The cannabinoid agonists anandamide (AEA) and CP55,940 both significantly augmented resting and KCl-evoked NPY release. AM251, a cannabinoid receptor antagonist, blocked the augmentation of NPY release elicited by AEA and CP55,940. Additionally, AM251 administered alone, in the absence of exogenous cannabinoid agonists, inhibited NPY release demonstrating the role of endogenous cannabinoids in NPY release. Combined, these findings demonstrate that cannabinoids augment NPY release in the hypothalamus and that this may be a potential mechanism behind the orexigenic activity of cannabinoids.