Catechol-O-methyltransferase (COMT) plays an important role in estrogen-induced cancers because COMT inactivates catechol estrogens that have cancer-promoting activities. Two promoters control the expression of human COMT isoforms: membrane-bound COMT (MB-COMT) and soluble COMT (S-COMT). We hypothesize that inactivation of MB-COMT and S-COMT is important in understanding the pathogenesis of endometrial cancer. To test this hypothesis, we investigated the methylation status and expression of two COMT isoforms in 4 endometrial cancer cell lines, 60 endometrial cancer tissues, 10 normal endometrium tissues from normal healthy controls, and 32 pairs of cancerous and normal endometrial samples from the same patients using methylation-specific PCR, methylation-specific sequencing, reverse transcription-PCR, and 5'-rapid amplification of cDNA ends. The results of this study clearly demonstrate that MB-COMT was inactivated and methylated, although S-COMT was activated and unmethylated in all endometrial cancer cell lines. The 5-aza-2'-deoxycytidine treatment restored MB-COMT expression in all cell lines. The promoter for MB-COMT was methylated in 47 of 60 cancer tissues but was unmethylated in endometrial tissues from cases without cancer. The promoter for S-COMT was unmethylated in all endometrial cancerous and normal tissues. The CpG methylation density at the MB-COMT promoter was significantly higher in cancer tissues (a mean of 79.1% of the 19 CpG sites; range, 69-94%) than in adjacent normal tissues (a mean of 8.7% of the 19 CpG sites; range, 3-14%). In summary, these findings demonstrate that methylation of multiple promoters of the COMT gene can selectively inactivate MB-COMT and may contribute to endometrial carcinogenesis.