RASSF1A is a major tumor suppressor gene located at 3p21.3. We investigated the role of aberrant promoter region hypermethylation of RASSF1A in a large series of adult gliomas. RASSF1A was frequently methylated in both primary tumors (36/63; 57%) and tumor cell lines (7/7; 100%). Hypermethylation of RASSF1A in glioma cell lines correlated with loss of expression and treatment with a demethylating agent-reactivated RASSF1A gene expression. Furthermore, reexpression of RASSF1A suppressed the growth of glioma cell line H4 in vitro. Next, we investigated whether other members of the RASSF gene family were also inactivated by methylation. NORE1B and RASSF3 were not methylated in gliomas, while NORE1A and RASSF5/ AD037 demonstrated methylation in glioma cell lines but not in primary tumors. We then investigated the methylation status of three other candidate 3p21.3 tumor suppressor genes. CACNA2D2 and SEMA3B were not frequently methylated, but the BLU gene located just centromeric to RASSF1 was frequently methylated in glioma cell lines (7/7) and in 80% (35/44) of glioma tumors. In these tumor cell lines, BLU expression was restored after treatment with a demethylating agent. Loss of BLU gene expression in glioma tumors correlated with BLU methylation. There was no association between RASSF1A and BLU methylation. RASSF1A methylation increased with tumor grade, while BLU methylation was seen at similar frequencies in all grades. Our data implicate RASSF1A and BLU promoter methylation in the pathogenesis of adult gliomas, while other RASSF family members and CACNA2D2 and SEMA3B appear to have only minor roles. In addition, RASSF1A and BLU methylation appear to be independent and specific events and not due to region-wide changes in DNA methylation.