Neuropeptide Y (NPY), a 36-amino-acid peptide, mediates biological effects by activating Y1, Y2, Y5, and y6 receptors. NPY neurons innervate many brain regions, including the hypothalamus, where NPY is involved in regulation of a broad range of homeostatic functions. We examined, by immunohistochemistry with tyramide signal amplification, the expression of the NPY Y2 receptor (Y2R) in the mouse brain with a newly developed rabbit polyclonal antibody. Y2R immunoreactivity was specific with its absence in Y2R knockout (KO) mice and in adjacent sections following preadsorption with the immunogenic peptide (10(-5) M). Y2R-positive processes were located in many brain regions, including the olfactory bulb, some cortical areas, septum, basal forebrain, nucleus accumbens, amygdala, hippocampus, hypothalamus, substantia nigra compacta, locus coeruleus, and solitary tract nucleus. However, colchicine treatment was needed to detect Y2R-like immunoreactivity in cell bodies in many, but not all, areas. The densest distributions of cell bodies were located in the septum basal forebrain, including the bed nucleus, and amygdala, with lower density in the anterior olfactory nucleus, nucleus accumbens, caudal striatum, CA1, CA2, and CA3 hippocampal fields, preoptic nuclei lateral hypothalamus, and A13 DA cells. The widespread distribution of Y2R-positive cell bodies and fibers suggests that NPY signaling through the Y2R is common in the mouse brain. Localization of the Y2R suggests that it is mostly presynaptic, a view supported by its frequent absence in cell bodies in the normal mouse and its dramatic increase in cell bodies of colchicine-treated mice.