Gene silencing via promoter hypermethylation is a central event in the pathogenesis of cancers. To identify novel methylation targets in colon cancer, we conducted a genome-wide, microarray-based, in silico, and epigenetic search. Complementary DNA microarray experiments were first performed to identify genes down-regulated in primary colon cancers and up-regulated in colon cancer cell lines after global DNA demethylation by 5-aza-2'-deoxycitidine. Candidate methylation targets were then identified by combining these microarray data with in silico genetic and functional searches. Candidate genes recognized by these searches were further investigated for promoter hypermethylation in colon cancer using methylation-specific polymerase chain reaction. We identified 51 novel and 3 known candidate methylation targets. Subsequent epigenetic analysis revealed that primary colon cancers demonstrated frequent methylation of somatostatin (SST, 30 of 34 cases, 88%) and the substance P precursor gene tachykinin-1 (TAC1; 16 of 34 cases, 47%). TAC1 methylation intensity was significantly higher in Dukes A/B than in Dukes C/D cancers (P = .01). SST methylation intensity was significantly higher in low-level microsatellite instability (MSI-L) than in non-MSI-L cancers (P = .02). Methylation was associated with messenger RNA down-regulation for both SST and TAC1. Furthermore, we isolated 5 additional novel promoter methylation targets: NELL1, AKAP12, caveolin-1, endoglin, and MAL. These data strongly suggest that SST and TAC1 are involved in colon carcinogenesis. Further studies are now indicated to elucidate mechanisms underlying their involvement in colon cancer and their values as clinical biomarkers. NELL1, AKAP12, caveolin-1, endoglin, and MAL are also promising tumor suppressor gene candidates deserving of further study.