Estrogen receptor (ER) beta gene codes for different transcript variants resulting from alternative splicing. In this study, we report identification of the two novel human exon-skipped ERbeta transcript isoforms ERbetaDelta125 and ERbetaDelta1256 in MDA-MD-231 breast cancer cells. Both transcripts could also be detected in a variety of human tissues. We further report the results of an in vitro attempt to characterize their function in regulation of cell growth, motility, apoptosis and gene expression. COS-1 cells stably transfected with the novel ERbeta transcripts exhibited a notably slower growth even in the absence of estradiol when compared to vector-transfected control cells. Like ERbeta1, both novel ERbeta transcript isoforms raised the basal apoptosis rate of COS-1 cells in a ligand-independent manner. Whereas introduction of ERbetaDelta1256 notably increased the sensitivity of COS-1 cells towards lower concentrations of selective estrogen receptor modulator tamoxifen, presence of ERbeta1 and ERbetaDelta125 transcripts further weakened the growth-inhibitory effect of tamoxifen on this cell line. Furthermore, expression of ERbetaDelta1256 variant was demonstrated to reduce transcript levels of estrogen-responsive genes like cyclin A2, IGFBP-4 and fibulin 1c in COS-1 cells in a ligand-independent manner. Though we were not able to detect the predicted 29 and 34kDa proteins by means of western blot analysis, our data strongly suggest the biological functionality of both isoforms on molecular level. With this report increasing the multitude of existing ERbeta mRNA isoforms, we provide further evidence that their synthesis has to be considered as an important level of estrogen signaling.