It has been shown in previous studies that a variety of estrogen receptor (ER) beta mRNA transcripts are expressed in human breast cancer cell lines and tumors. To complement the RNA expression studies, we have developed ER-beta-specific antibodies to characterize ER-beta protein expression in breast cancer cell lines and tumors. Monoclonal antibodies were made against a peptide representing the first 18 amino acids of the longest ER-beta open reading frame reported to date, and polyclonal antibodies were made against a peptide within the ER-beta B domain. By Western blot analysis, we show that ER-beta protein is expressed in all cancer cell lines tested and in three of five breast tumor samples. The breast cancer cell lines showed variation in the size of the expressed ER-beta protein. The longest form detected was consistent with the 530-amino acid, full-length ER-beta sequence. Shorter ER-beta isoforms were detected in the ER-alpha-negative MDA-MB-231 and MDA-MB-435 breast cancer cell lines, likely corresponding to previously described COOH-terminal RNA variant isoforms.