2-Methoxyestradiol (2ME), a metabolite of estradiol (E), inhibits proliferation of various tumor cells. In this study we determined the effect of 2ME on human glioblastoma cell lines, in vitro. We compared these cells with cultured astrocytes obtained from traumatized adult rat striatum. Exposure to 2ME had a strong antiproliferative effect on human glioblastoma and caused an increase in the population of apoptotic cells, detected by flow cytometry, in some of the investigated cell lines. A significant number of cells were blocked in the G2/M phase of the cell cycle. Concurrently, the population of cells in the G1 phase decreased in all glioblastoma cell lines. Staining with Hoechst 33258 revealed abnormal nuclear morphology in the proliferating cells treated with 2ME. Treatment with 2ME induced upregulation of wild type p53 in one of the human glioblastoma cell lines as well as in proliferating adult rat astrocytes. We conclude that 2ME inhibits the growth of human glioblastoma cell lines and induces apoptosis, in vitro. This compound deserves further investigation as a treatment for gliomas.