Previous studies have suggested an important role of ERs (oestrogen receptors) in the pathogenesis of leukaemias. However, there is no information so far about the epigenetic characteristics of ERalpha isoforms and ERbeta in leukaemias. In the present study, the mRNA expression and promoter CpG methylation of ERalpha isoforms (i.e. ERalpha-A, -B and -C) and ERbeta in leukaemia cell lines were evaluated using RT-PCR (reverse transcription-PCR) and MSP (methylation-specific PCR) respectively. The methylation of ERs was further analysed in acute leukaemia patients by MSP and direct DNA sequencing. Although all ERalpha isoforms and ERbeta were methylated in all leukaemia cell lines, except for ERalpha-C, which was unmethylated in HL-60 and K562 cell lines, only the expression of ERalpha-A was deficient in all cell lines and its expression could be reactivated by DNA demethylation reagents. With regard to the methylation characteristics in acute leukaemia patients, only ERalpha-A was inactivated and specifically methylated (95%; 38/40) in almost all patients and unmethylated in all healthy controls, whereas ERalpha-B, -C and ERbeta were methylated in both patients and healthy controls. This result suggested that the methylated status of ERalpha-A might serve as an epigenetic biomarker of leukaemias. The present study is the first report that demonstrates selective inactivation of ERalpha isoforms through the promoter CpG methylation pathway in leukaemias.