Multiple transcripts which arise from the human estrogen receptor beta (ER beta) gene have been characterized. Three full length isoforms of the hER beta gene, designated hER beta 1-3, were identified in a testis cDNA library. An additional two isoforms, designated hER beta 4 and hER beta 5, were identified by PCR amplification from testis cDNA and from the MDA-MB 435 cell line. hER beta 1 corresponds to the previously described hER beta. All five isoforms diverge at a common position within the predicted helix 10 of the ligand binding domain of hER beta, with nucleotide sequences consistent with differential exon usage. The hER beta isoform mRNAs displayed a differential pattern of expression in human tissues and in tumor cell lines when analyzed by RT-PCR. Further characterization of the three full length isoforms, hER beta 1-3, by in vitro band shift studies indicated that the isoforms were able to form DNA-binding homodimers and heterodimers with each other and with the ER alpha subtype.