Estrogen receptor alpha (ER-α) is an important regulator of growth and differentiation in the mammary gland and the female reproductive tract. It is also involved in the development of malignant tumors. The human ER-β is highly homologous to the extensively studied human ER-α. It binds to the endogenous 17β-estradiol with similar affinity as ER-α and the transcriptional activity through the consensus ERE can be stimulated. Five ER-β isoforms were cloned and characterized. They diverge at a common position within the predicted helix 10 of the ligand-binding domain of the human ER-β, with nucleotide sequences consistent with different exon usage. These isoforms of human ER-β show differential expression in tissues and in tumor cell lines. Furthermore, they are predicted to form DNA-binding heterodimers when coexpressed. Expression of some of the ER-β isoforms in human breast tissue, breast cancer, and breast cancer cell lines were reported by several groups. However, there is no complete analysis of the gene expression pattern of all ER-β isoforms in breast cancer so far. In this study, we examined the mRNA expression of each of the ER-β isoforms in 30 tumors from breast cancer patients and 21 breast cell lines. In conclusion, expression of ER-β1, ER-β2, and ER-β5 were observed in different cell lines as well as in the tumors, ER-β4 isoform was expressed in all samples, and ER-β3 isoform was not detected in any of the samples examined. There were no associations of the expression of all ER-β isoforms with the invasiveness of the cell lines as well as with clinical parameters of the tumors.