Methylation of scattered CpG sites within a CpG island (CGI), denoted as "methylation seeds", is proposed to serve as a precursor of dense methylation of the CGI. We examined whether or not an abnormal increase of methylation seeds is associated with a larger number of methylation-silenced genes. Two gastric cancer cell lines with increased seeds (AGS and KATO-III) and two cell lines without (HSC39 and HSC57) were treated with equivalent doses of a demethylating agent, 5-aza-2'-deoxycytidine. Oligonucleotide microarray analysis showed that 25, 63, 9 and 1 genes with promoter CGIs, respectively, were up-regulated at 16-fold or more. By methylation analysis, it was estimated that 24, 41, 4 and 1 gene, respectively, were silenced due to methylation of promoter CGIs. Notably, AGS and KATO-III had silencing of genes expressed in the normal stomach while HSC39 and HSC57 had few. The association between the increase of methylation seeds and larger numbers of silenced genes suggested that the increase can induce gene silencing overriding their transcription.