The Mxi1 protein functions in a regulatory network with members of the c-Myc family, in which c-Myc activates transcription and stimulates cell proliferation, and Mxi1 negatively regulates these actions. Inactivation of the MXI1 gene could, therefore, inhibit differentiation and enhance proliferation in the presence of normal levels of c-Myc, and thus MXI1 is a potential tumor suppressor gene. We and others have previously mapped the MXI1 gene to the distal portion of chromosome 10q, a region that is rearranged or affected by allelic loss in many astrocytic brain tumors. Using a newly described polymorphic CA microsatellite repeat in the third MXI1 intron, we show that 7 of 11 informative glioblastomas demonstrated MXI1 allelic loss. Sequence analysis revealed no somatic mutations in any of the six MXI1 coding exons, similar to findings in prostate tumors with MXI1 allelic loss. To determine whether MXI1 can indeed function as a suppressor of growth, we have introduced a steroid-inducible MXI1 expression vector into the U87MG cell line, a glioblastoma cell line lacking endogenous MXI1 expression. Induction of MXI1 expression resulted in a decreased growth rate and distinct morphological changes. Furthermore, cell cycle analysis demonstrated that induction of MXI1 results in accumulation of cells in the G2-M phase. Thus, these studies support the notion that MXI1 normally functions to suppress cell growth and suggest that loss of MXI1 function may play a role in human glioblastoma development.