BACKGROUND: Expression and transcriptional activity of genes are regulated byseveral factors, including DNA methylation. We examined the frequency of DNA hypermethylation at two nucleotide positions, the proximal promoter region (PPR) in exon 1 and the distal promoter region (DPR) in exon 1' of the estrogen-receptor alpha (ER alpha) gene in human breast cancer, and the correlation between ER and progesterone receptor (PgR) status. METHODS: The frequency of hypermethylation of PPR and DPR in 124 breast cancerswas examined by the semiquantitative competitive polymerase chain reaction (COM-PCR) assay with restriction enzymes. ER and PgR proteins were analyzed by enzyme immunoassay (EIA; fmol/mg) to determine whether DNA hypermethylation influences the status of either protein. RESULTS: There were no significant differences in ER protein status between DNAmethylated and unmethylated groups for either PPR (71.2 +/- 190.1 versus 60.7 +/- 88.4) or DPR (70.0 +/- 183.6 versus 60.9 +/- 89.3). There was a significant differencein PgR protein status between these two groups for PPR (46.8 +/- 67.1 versus 169.1 +/- 394.9, P< 0.01). When one positional methylation was regarded as the criterion for hypermethylation, the frequency of hypermethylation in the ER(+)PgR(-)phenotype was significantly higher than in the ER(+)PgR(+), ER(-)PgR( +) and ER(-)PgR(-) phenotypes (72.7% versus 31.3%, 40.0% and 28.6%, P< 0.01). CONCLUSION: Hypermethylation of the 5'-upstream side of the ER alpha gene did notcorrelate with lack of ER, but did correlate with lack of PgR, and particularlywith the expression of the ER(+)PgR(-) phenotype. We conclude that DNA hypermethylation of PPR and DPR in the Er alpha gene reflects the expression of the ER target gene rather than the ER gene itself and may account for anti-estrogen resistance in ER-positive breast cancer.