Human chorionic gonadotropin (hCG) suppresses cell-mediated allogeneic reactions, viral replication, tumorigenesis, and metastasis, most of which require activation of nuclear transcription factor-kappaB (NF-kappaB) and activator protein-1 (AP-1). In the present report, we investigated the effect of hCG on NF-kappaB and AP-1 activated by tumor necrosis factor (TNF). Treatment of the CaCOV3 human ovarian cell line with hCG blocked TNF-induced activation of NF-kappaB, IkappaBalpha degradation, and NF-kappaB-dependent reporter gene transcription. hCG also blocked NF-kappaB activation induced by ceramide. The effect of hCG on NF-kappaB was mediated through inhibition of phosphorylation of IkappaBalpha. Because hCG also blocked TNF receptor-associated factor-2 and NF-kappaB-inducing kinase reporter gene expression, hCG must act at a step that causes phosphorylation of IkappaBalpha. AP-1 activation induced by TNF and ceramide was also suppressed by hCG. hCG abrogated the TNF-induced activation of mitogen-activated protein kinase kinase and c-Jun N-terminal kinase required for NF-kappaB and AP-1, respectively. Dideoxyadenosine and H-8 reversed the effect, and dibutyryl cAMP mimicked the effect, suggesting that hCG suppresses the transcription factors through cAMP-induced protein kinase A pathway. Overall, our results indicate that hCG inhibits the activation of NF-kappaB and AP-1, which may be the molecular basis by which hCG suppresses viral replication, cell proliferation, tumorigenesis, and metastasis.