Demethylation of specific sites or restricted genomic regions has been reported to correlate with gene activation and also with carcinogenesis. As abnormal expression of Estrogen Receptor (ER) could be involved in the genesis or progression of tumors in estrogen target tissues, the methylation of ER gene has been compared in 8 endometrial carcinomas and 29 normal endometria. In order to look for a correlation between methylation and expression, levels of ER RNA were also measured. While the 5' region of ER gene was found to be demethylated in both normal and carcinomatous tissues, there was demethylation of some specific sites in the internal part of the gene only in the carcinomas examined. In addition, in the carcinomatous tissues the levels of ER RNA were low, indicating that an increase of ER gene hypomethylation does not raise, and even may reduce, the ER expression in endometrium. The abnormal undermethylation observed in ER gene appears to be unrelated to general DNA hypomethylation which is frequently present in neoplastic tissues; nor has it been found in ER DNA isolated from breast carcinomas. These data strongly support the hypothesis that such a methylation is specifically related to endometrial transformation and therefore it can be considered an additional marker of this disease.