The estrogen receptor (ER) expression and HER2 amplification are important factors in determining the prognosis and therapy of breast cancer. Interactions between the two signaling pathways for example resulted in ERalpha-dependent regulation of HER2 expression in breast cancer cells. In this study, we investigated to what extent ERbeta is able to affect the HER2 expression. For this purpose, we analyzed HER2 levels in ERbeta1-overexpressing clones of the breast cancer cell lines MCF-7 and SK-BR-3 and of the ovarian cancer cell lines SK-OV-3 and OVCAR-3 by both RT-PCR and Western blot analysis. Treatment with ligand 17-beta estradiol diminished the HER2 expression in MCF-7 wild-type cells, an effect partially inhibited by treatment with 4-OH tamoxifen. MCF-7 breast cancer cells stably overexpressing ERbeta1 exhibited elevated >5-fold HER2 mRNA levels and elevated >3-fold HER2 protein levels even in the absence of estradiol. In contrast, ERbeta1 overexpression did not affect HER2 protein levels in the ERalpha-positive OVCAR-3 ovarian cancer cells and in the HER2 overexpressing, hormone-independent SK-BR-3 and SK-OV-3 cells. By demonstrating the elevated HER2 expression in a hormone-dependent breast cancer cell line overexpressing ERbeta1, our data suggest the presence of cross-talk between the two receptors. This is one of the molecular mechanisms underlying the significant ERbeta/HER2 co-expression observed in recent clinical studies.