Oestrogen receptor alpha (ER-alpha) gene has two specific promoters, distal (P0) and proximal (P1), which induce almost identical transcripts in size due to different splicing. We examined the methylation at both promoter regions of the ER-alpha gene using HpaII, a methylation-sensitive restriction enzyme, prior to polymerase chain reaction (PCR) amplification. To confirm the results of PCR-based methylation analysis, Southern hybridization was also performed. Twenty of 29 patients with ER-alpha-positive tumours and five of 27 with ER-alpha-negative tumours were unmethylated at the P1 promoter region of the ER-alpha gene. The incidence of methylation was highly negatively correlated with ER-alpha expression (P = 0.0002). A similarly negative correlation was observed at the P0 promoter region of the ER-alpha gene (P = 0.0154). Additionally, the tumours with the ER-alpha gene hypermethylated at both promoter regions had definitely negative ER-alpha values. It was suggested that this epigenetic change might control ER-alpha expression, and might play an important role in the loss of hormone-dependence in breast cancer.