2076 Background: Inhibitors of DNA binding/differentiation (ID1-4) are a family of helix-loop-helix transcription factor, highly expressed during embryogenesis and at lower levels in mature tissues. Expression of ID proteins have related to features of malignancies, including cellular transformation, angiogenesis and metastasis. ID4 plays an important role in neuronal stem cell differentiation and its deregulation has been implicated in glial neoplasia. Recent works have shown that hypermethylation of ID4 can determine the silencing of this protein in several human cancers. Methods: We analyzed the methylation status of ID4 by methylation-specific-PCR in 85 glioblastoma multiforme (GBM) cases and in 20 normal brain tissues. After surgical treatment, all GBMs were subjected to a cycle of radiotherapy (2 Gy per fraction, once a day, 5 days a week, 60 Gy total dose) with concomitant administration of temozolomide (75 mg per square meter of body surface area per day) for 7 days a week from the first to the last day of radiotherapy, followed by 5 cycles of adjuvant temozolomide (at 200 mg per square meter of body surface area on days 1 through 5) given at four-week intervals. Methylation status was confirmed by sequencing after subcloning and RNA and protein expression was assessed by real-time PCR and immunohistochemistry. The relationship between ID4 methylation and clinical outcome was investigated. Results: The promoter of ID4 was methylated in 17/85 GBMs. After subcloning, the methylation was confirmed by sequencing in over 95% of GpC dinucleotides. In methylated GBMs, ID4 mRNA expression was reduced by 5 folds as compared with unmethylated GBMs. A significant reduction of protein expression was detected in all hypermethylated cases. ID4 methylation was significantly associated with a favourable clinical outcome (p=0.0033). No significant differences were found between patients with and without ID4 methylation with respect to age, sex, p53 mutation, Ki67 labeling index and extent of surgical resection. Conclusions: Our data suggest that methylation of ID4 may be involved in the pathogenesis of GBM and in the resistance of this neoplasm to conventional treatment.