Granulosa cell tumors of the ovary (GCT) are a distinct, hormonally active subset of ovarian cancers. Although it has recently been shown that approximately 97 of all adult GCT harbor a novel somatic missense mutation in the FOXL2 gene, given its almost universal presence, it does not explain differences in tumor stage and/or recurrence. The nuclear factor kappaB (NFkappaB) transcription factor is constitutively active in two human GCT-derived cell lines, COV434 and KGN, which are useful in vitro models to investigate juvenile and adult GCT, respectively. This study aimed to determine the molecular basis and pathogenetic significance of this aberrant NFkappaB activity. Selective chemical inhibitors were used to target candidate components of the pathway. The constitutive activity was blocked by two independent inhibitors of IkappaBalpha phosphorylation, suggesting that aberrant activation occurs upstream of this point. NFkappaB inhibition resulted in a dose-dependent decrease in cell proliferation and viability and a dose-dependent increase in apoptosis. Inhibitors of earlier components of the pathway were without effect. Two independent inhibitors of inhibitor of kappaB kinase (IKK)beta, a catalytic subunit of the NFkappaB activation complex, were unable to inhibit the constitutive activity, but surprisingly also ligand-induced activity. These findings suggest a central role for IKKbeta; however, no mutations or altered expression of the IKKbeta, IKKalpha, or IKKgamma genes was observed in the cell lines or in a panel of human GCT samples. This study highlights unresolved issues in understanding the pathogenesis of GCT and in the use of the COV434 and KGN cells lines as model systems.