A triple-primer PCR assay was developed, based on the coamplification of estrogen receptor (ER)-beta1, -beta2, and -beta5 cDNAs, to investigate the relative expressions of the corresponding mRNAs in breast cancer lines and in 53 independent breast tumors. The expression of ER-beta2 and ER-beta5 mRNAs was higher than that of ER-beta1 mRNA in both cancer cell lines and breast tumors. In breast tumors, increases in the ER-beta2:ER-beta1 and ER-beta5:ER-beta1 mRNA expression ratios were observed, which positively correlated with the level of tumor inflammation and tumor grade, respectively. A trend toward an increase of these ratios was also found in tumors, as compared to the normal adjacent breast tissue available for 13 cases. Our data suggest that changes in the relative expression of ER-beta1, -beta2, and -beta5 mRNAs occur during breast tumorigenesis and tumor progression.