Inactivation of specific tumor suppressor genes by transcriptional silencing associated with hypermethylation of the promoter is a common event in cancer. We have applied the amplification of intermethylated sites (AIMS) technique to a 100 human colorectal cancers and seven cell lines to identify recurrent alterations that may unveil silenced tumor suppressor genes. Bisulfite sequencing was used to confirm differential DNA methylation results. Gene expression analysis was performed by real-time RT-PCR. An AIMS band recurrently displayed in tumors but not in normal tissues was isolated and identified as part of the CpG island of the prostacyclin synthase (PTGIS) gene promoter. PTGIS promoter was hypermethylated in 43 out of 100 colorectal cancers and in all cell lines. Bisulfite sequencing and clonal analysis confirmed the results obtained by AIMS and demonstrated biallelic hypermethylation of PTGIS promoter. Hypermethylation of the PTGIS promoter was associated with diminished gene expression, that was restored after treatment with demethylating and histone deacetylases inhibitor agents. PTGIS hypermethylation was associated with aneuploidy and p53 mutations. In the adjusted model, PTGIS methylation, but not p53 mutation, maintained the association with aneuploidy. We conclude that epigenetic inactivation of the PTGIS gene is a recurrent alteration in colorectal carcinogenesis.