It has been noted that the expression of Sd(a), including its antigenic structure, the beta-1,4-N-acetylgalactosyltransferase II (beta4GalNAcT-II) activity responsible for its formation, and the Sd(a) beta4GalNAcT-II mRNA transcript, is drastically reduced in oncogenetic processes in gastrointestinal tissues. Markedly reduced metastatic potential has been demonstrated in colon and gastric cancer cells transfected with the Sd(a) beta4GalNAcT-II gene. In this study, a putative CpG island encompassing the promoter and exon 1 regions in the human Sd(a) beta4GalNAcT-II gene was identified, and the investigation of DNA methylation of the Sd(a) gene promoter region demonstrated a clear association between the methylation status of the CpG island promoter and expression of the Sd(a) gene in gastrointestinal cancer cell lines. Hypomethylation of the promoter region of the Sd(a) gene was shown in cells where this gene was expressed. By contrast, there was significant hypermethylation of the Sd(a) gene promoter in cells that did not express the gene. A specific methylation profile in the Sd(a) gene CpG island was demonstrated in KATO III gastric cancer cells. In colon cancer cells with the hypermethylated Sd(a) gene promoter, treatment with the DNA methylation inhibitor, 5-aza-2'-deoxycytidine, resulted in demethylation of the promoter region and substantially induced the expression of the Sd(a) gene and the Sd(a) antigenic structure. These results strongly suggest that promoter DNA methylation plays a crucial role in the regulation of the Sd(a) beta4GalNAcT-II gene and Sd(a) antigen expression.