Estrogen receptor (ER) is the predominant estrogen receptor in granulosa cells of the ovary. ER 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 ER 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 co-transfected ER or . This transcriptional resistance is specific to steroid receptor transactivation; reporter plasmids that are activated by the transcription factors AP-1 and NF-B demonstrate both constitutive and inducible transactivation. AP-1 and NF-B are known to cause transrepression of both ER and GR 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-B. The AP-1 inhibitors alone had no effect, whereas inhibition of NF-B signaling allowed a 3-4 fold estradiol-mediated induction of ERE₂-luc. This response was ligand- and ER-dependent. Repression of ER signaling by NF-B has not previously been reported. Recent evidence suggests that ER may function to promote differentiation. The inhibition of ER in combination with the anti-apoptotic properties of NF-B may therefore contribute to pathogenesis of GCT.