Five isoforms of estrogen receptor beta (ER-B) have been cloned, and were shown to have different amino acid sequences at their ligand binding domains. The aim of this study was to determine the protein expression of ER-B isoforms in human breast cancer tissues. Sections of formalin-fixed and paraffin-embedded tissue from 17 invasive breast carcinomas were immunostained for ER-B1, 2, 3 and 5 using affinity purified antibodies and the immunoperoxidase technique. ER-B1, 2, 3 and 5 expression in cancer cells was variable, with both nuclear and cytoplasmic staining seen. ER-B1 and ER-B2 were the most commonly expressed, whereas ER-B3 was uncommon. ER-B3 and ER-B5 expression was associated with larger tumor size and higher proliferative activity, but only ER-B3 expression was associated with lymph node metastasis. ER-B1, 2 and 5 were detected in normal epithelial cells; ER-B1 and 2 in endothelial, stromal, and myoepithelial cells; and ER-B2 in nerves. Our findings show that ER-B isoforms are differentially expressed in breast cancer cells and in benign epithelial and non-epithelial components of breast tissue, and suggest that ER-B isoform specific agonists and antagonists are likely to have different biological effects on normal and cancerous cells and tissues.