AbstractCpG‐island hypermethylation of gene promoters is a frequent mechanism for gene inactivation in tumors. Many neuroblastomas have hypermethylation and down‐regulation of CASP8, leading to resistance to tumor necrosis factor–related apoptosis‐inducing ligand (TRAIL). We recently found hypermethylation of the four TRAIL receptors in 9 neuroblastoma cell lines. Here, we analyzed methylation of 34 genes in 22 neuroblastoma cell lines. Of the 29 newly analyzed genes, only FLIP at chromosome band 2q33 was methylated in 8/22 cell lines. The FLIP protein is a negative regulator of Caspase 8. FLIP maps adjacent to CASP8, and their methylation patterns showed a moderate correlation. Furthermore, co‐methylation patterns were observed for the TRAIL receptor pairs DCR1 and DCR2 and between DR4 and DR5. All four receptors co‐localize in chromosome band 8p21. The 6 genes methylated in neuroblastomas appeared to occur in pairs. The genes within each pair have a strong sequence homology and originated from gene duplication. We found no evidence for regional spreading of methylation, given that we did not observe de novo methylation in additional local CpG islands. However, the gene pairs showed a striking co‐regulation at the mRNA expression level. Down‐regulation of FLIP strongly corresponds with down‐regulation of CASP8, and this was also found for DCR1 and DCR2. Only a subset of the down‐regulated genes was methylated. This suggests a mechanism of co‐regulated transcriptional silencing of the gene pairs, followed by a methylation event that is less penetrating. The methylation pattern therefore supports a model in which CpG islands are not randomly targeted by methylation in cancer. Specific transcriptional silencing probably marks genes that can become methylated. © 2003 Wiley‐Liss, Inc.