The phthalates are ubiquitous industrial plasticizers and include agents such as di(2-ethylhexyl) phthalate (DEHP), dibutyl phthalate (DBP), and butyl benzyl phthalate (BBP), which are classified as endocrine disruptors because of their anti-androgenic or pro-estrogenic effects. A recent study suggested that DBP produced the hypomethylation of c-myc protooncogene in mouse liver to activate c-myc. In the present study, DNA hypomethylation at the promoter region of the estrogen receptor alpha (ERalpha) gene was investigated by methylation-specific polymerase chain reaction (MSP) in a human breast cancer (MCF7) and in a normal (MCF10A) cell line after DBP treatment. Yeast-based estrogen receptor transcription assays showed that hERalpha gene expression was induced by BBP but not DBP. Moreover, MCF7 cells treated with BBP or DBP at 10(-5)M led to the demethylation of ERalpha promoter-associated CpG islands. These data suggest that an altered ER mRNA expression by BBP might be related to aberrant DNA methylation in the promoter region of ERalpha.