Methylation of promoter-associated CpG islands appears to be a potential way by which tumor suppressor genes are inactivated in cancer. Using Southern blot analysis, we have studied the methylation of several genes in glioblastoma multiforme (GBM), trying to determine their contribution to tumorigenesis. Genes studied included the estrogen receptor (ER), N33, the candidate tumor-suppressors P15, P16 and HIC1 and a control gene, c-abl. Hypermethylation of N33, ER, HIC1, P16, P15 and c-abl were found in 61%, 59%, 60%, 5%, 2% and 0% of GBM respectively. HIC1 methylation was detected in normal brain as well, but appeared to be more extensive in tumors. ER and N33 methylation were significantly more frequent in tumors from individuals over the age of 40 (70% and 88% vs 36% and 14%). In addition, there was a strong association between ER and N33 methylation, which were concordant in 81% of the cases (P<0.01). ER and N33 methylation in GBM may therefore appear as a result of shared etiologic factors, which may relate in part to aging cell populations in the brain.