We evaluated the significance of aberrant DNA methyltransferase expression in human carcinogenesis by examining 32 colorectal and 34 stomach cancers. Levels of mRNAs encoding DNA methyltransferases were measured by reverse transcription, followed by real‐time quantitative detection of PCR products. The DNA methylation state of CpG islands and peri‐centromeric satellite regions was examined by bisulfite modification and Southern blotting, respectively. The average level of mRNA for DNMT1 and DNMT3b in colorectal and stomach cancers was significantly higher than in corresponding non‐cancerous mucosae, whereas the average level of mRNA for DNMT2 was significantly lower in colorectal and stomach cancers than in non‐cancerous tissue. Over‐expression of DNMT3b in stomach cancer was significantly higher in cases with lymph node metastasis than in cases without. DNA hypermethylation on the p16, human Mut L homologue‐1 and thrombospondin‐1 genes and the methylated in tumor (MINT) 1, 2, 12, 25 and 31 clones was found in 23%, 27%, 9%, 23%, 20%, 23%, 20% and 10% of the colon cancers and in 9%, 19%, 30%, 25%, 34%, 19%, 81% and 3% of the stomach cancers, respectively. Criteria for identification of the CpG island methylator phenotype (CIMP) were met in 23% of colorectal cancers and 31% of stomach cancers. DNA hypomethylation on satellites 2 and 3 was detected in 0% and 8% of colorectal and stomach cancers, respectively. Over‐expression of DNMT1 mRNA was significantly associated with CIMP, whereas the level of DNMT3b mRNA was not associated with CIMP or DNA hypomethylation of peri‐centromeric satellite regions. These data suggest that both over‐expression of the maintenance DNA methyltransferase DNMT1 and over‐expression of a newly identified de novo DNA methyltransferase, DNMT3b, are involved in human carcinogenesis, probably at different stages and through different mechanisms. © 2001 Wiley‐Liss, Inc.