A key issue in the development of the central nervous system (CNS) is understanding the molecular mechanisms regulating cell number. The present study examines the role of CD81 (previously known as TAPA, the target of the antiproliferative antibody) in the control of brain size and glial cell number. CD81 is a member of the tetraspanin family of proteins. This group of small membrane proteins is associated with the regulation of cell migration and mitotic activity. Glial cells express CD81, and antibodies directed against this protein suppress the mitotic activity of cultured cells. In this study, we examine the effects of the CD81 −/− mutation on the CNS of mature mice. These mice have extremely large brains, as much as 30% larger than the brains of wild‐type (+/+) littermates. The increase in brain weight is accompanied by an increase in the number astrocytes and microglia, whereas the number of neurons and oligodendrocytes in the CD81 −/− animals appears to be normal. When the CD81 −/− mutation is placed on different genetic backgrounds, there is a remarkable range in the penetrance of the null allele phenotype, demonstrating that the mutation can be affected by modifier loci. This work provides support for the role of CD81 in the regulation of astrocyte and microglial number, perhaps by regulating cell proliferation by a contact inhibition‐dependent mechanism. J. Comp. Neurol. 453:22–32, 2002. © 2002 Wiley‐Liss, Inc.