Estrogen receptor (ER) β is expressed in a number of tissues, including the breast. We have recently shown that ER-β mRNA is regulated by estradiol (E2) and that antiestrogens antagonize E2 induction of ER-β mRNA. Here, we identify by reverse transcription-PCR and by the RNase protection assay a mRNA coding for a variant of ER-β that is coexpressed with wild-type ER-β in the ER-α-negative, estrogen-independent breast cancer cell line MDA-MB-231 and in malignant breast tumor specimens. In contrast, this variant was not seen in the tested normal breast tissue. Sequence analysis of the ER-β variant PCR product revealed the absence of 139 bp within the hormone-binding domain. This ER-β deletion corresponds precisely to the entire exon 5 of ER-α. The ER-β variant protein is predicted to lack part of the hormone-binding domain and may bind E2 with lower affinity than the wild-type ER-β protein.