The peptide transmitter neuropeptide Y (NPY) has been implicated in a plethora of actions in the central nervous system, including the hippocampus and neocortex (NeoCx). Previous studies using traditional receptor autoradiography show that NPY receptor binding is altered under various pathophysiological conditions. However, these do not provide information about downstream signal transduction. To this means, agonist-stimulated [³⁵S]GTPγS binding has in recent years been introduced as a method for measuring receptor activation of intracellular G-proteins. In the present study, this method was further optimized to visualize the distribution of individual NPY receptor-mediated [³⁵S]GTPγS binding in the mouse hippocampus and NeoCx using the endogenous ligand NPY in combination with optimized concentrations of selective antagonists for Y1, Y2, and Y5 NPY receptors. Consistent with previous studies, Y1 receptor-mediated [³⁵S]GTPγS binding was mainly found in the dentate gyrus and NeoCx while Y2 receptor-mediated [³⁵S]GTPγS binding predominated in the hippocampal CA3 and CA1. Only very low levels of Y5 receptor-mediated [³⁵S]GTPγS binding appeared to be present in the hippocampus and NeoCx. Furthermore, the effect of NPY receptor antagonists per se was also studied. Both BIIE0246 and L-152,804 significantly attenuated the basal [³⁵S]GTPγS binding response, suggesting inverse agonism.