An alternatively spliced isoform of human estrogen receptor beta (ER beta) has been isolated from normal human testis mRNA that is coexpressed with wild-type ERP by reverse transcription polymerase chain reaction (RT-PCR). Sequence analysis of the ER beta isoform PCR product reveals the absence of 139 bp that corresponds to the entire exon 5 of wild-type ER beta, which predicts 60 lack part of the hormone-binding domain, The transient expression of the exon 5-deleted isoform of ERP (ERP beta Delta5) had no effect on basal transactivation activity of an estrogen-responsive luciferase reporter gene. This finding was in contrast to the previous reports that the exon 8-deleted isoform of ER alpha (ER alpha Delta5) acts as a dominant positive receptor, increasing basal gene transactivation itself. Moreover, when ER beta Delta5 was cotransfected with the wild-type ER alpha or ER beta, it behaved as a dominant negative receptor that inhibited not only estradiol-stimulated transactivation by ER beta but also that by ER alpha. The ligand-independent nuclear localization of ER beta Delta5 was confirmed by immunohistochemistry, and the coexpression of the isoform and the wild-type receptors could be observed in a single cell that transfected with both receptor cDNAs, These findings indicate that ER beta Delta5 has a potential as a dominant negative receptor that blocks both ER alpha and ER beta signaling pathways, suggesting some physiological roles of this isoform as an "ER inhibitor''.