Neuropeptide Y (NPY) is one of the most abundant peptides in the central nervous system. Its effects on, for example, cognition, memory and motor functions are thought to be mediated mainly via its interactions with the NPY Y1 and Y2 receptor subtypes. We had previously described the neuroanatomical organization of the Y1 and Y2 mRNA expression in humans. However, in view of the lack of information regarding the overall detailed distribution of NPY mRNA expression in the human brain, a complete picture of the anatomical organization of the NPY-related genes was still missing. Thus, in the present study, the regional distribution of NPY mRNA-expressing cells was analyzed in the post-mortem human brain. In addition, double labeling in situ hybridization was performed to characterize the NPY neuronal populations in relation to the Y1 and/or Y2 receptor mRNA localization in the human cerebral cortex, striatum, and amygdala. NPY mRNA was found to be abundant in layers II and VI of the neocortex, polymorphic layer of the dentate gyrus, basal ganglia, and amygdala. Double labeling in situ hybridization showed the co-expression of NPY mRNA with the Y2, but not with the Y1, mRNA in the human cerebral cortex, hippocampus, amygdala, striatum, and nucleus accumbens, and the existence of co-expression of the Y1 and Y2 mRNAs in the cerebral cortex and amygdala. Overall, these results suggest a role for the Y2, but not Y1, as an autoreceptor in the NPY neuronal populations of the human brain.