Numerous studies have suggested that astrocytes in the central nervous system (CNS) exhibit molecular and functional heterogeneity. In this regard, astroglia from different CNS locations express distinct immune system, and neurotransmitter proteins, have varying levels of gap junction coupling and respond differently to injury. However, the relevance of these differences to human disease is unclear. As brain tumors in children arise in specific CNS locations, we hypothesized that regional astroglial cell heterogeneity might partly underlie the propensity for gliomas to arise in these areas. In this study, we performed high-density RNA microarray profiling on astrocytes from postnatal day 1 optic nerve, cerebellum, brainstem, and neocortex. We showed that astroglia from each region are molecularly distinct, and we were able to develop gene expression patterns that distinguish astroglia, but not neural stem cells, from these different brain regions. We next used these microarray data to determine whether brain tumor suppressor genes were differentially expressed in these distinct populations of astroglia. Interestingly, neurofibromatosis type 1 (NF1) gene expression was decreased at both the RNA and protein levels in neocortical astroglia relative to astroglia from the other brain regions. To determine the functional significance of this finding, we found increased astroglial cell proliferation in optic nerve, brainstem, and cerebellum, but not neocortex, following Nf1 inactivation in vitro and in vivo. These findings provide molecular evidence for CNS astroglial cell heterogeneity, and suggest that differences in tumor suppressor gene expression might contribute to the regional localization of human brain tumors. © 2009 Wiley-Liss, Inc.