Estrogen receptor (ER) β is the predominant ER in granulosa cells of the ovary. ERβ is expressed at high levels in granulosa cell tumors (GCT) and in the human GCT-derived cell lines, COV434 and KGN. To gain insight into ERβ function in granulosa cells and in GCT, we have used the COV434 and KGN cell lines. Although the cells bind estradiol (E2), transcriptional activation of a transfected estrogen-responsive reporter vector construct (ERE2-luc) by E2 was not observed. Transactivation was also not observed with cotransfected ERα or β. This transcriptional resistance is specific to steroid receptor transactivation; reporter plasmids that are activated by the transcription factors activator protein 1 (AP-1) and nuclear factor κB (NF-κB) demonstrate both constitutive and inducible transactivation. AP-1 and NF-κB are known to cause transrepression of both ERα- and glucocorticoid receptor-mediated transcription. We therefore examined the possibility that activation of these pathways was responsible for the lack of a response to estrogen by using inhibitors of AP-1 or NF-κB. The AP-1 inhibitors alone had no effect, whereas inhibition of NF-κB signaling allowed a 3- to 4-fold E 2-mediated induction of ERE2-luc. This response was both ligand and ER dependent. Repression of ERβ signaling by NF-κB has not previously been reported. Recent evidence suggests that ERβ may function to promote differentiation. The inhibition of ERβ in combination with the antiapoptotic properties of NF-κB may therefore contribute to pathogenesis of GCT.