The HIV-1 long terminal repeat (LTR) responds to a variety of cellular signal transduction pathways. We demonstrate that the cAMP-dependent protein kinase A (PKA) and protein kinase C (PKC) signaling pathways synergize to increase HIV-1 LTR-mediated transcription and viral replication in a latently infected promonocytic cell line (U1). The LTR-mediated synergy induced by cholera toxin (Ctx), a potent activator of the cAMP-dependent PKA pathway, and the PKC activator phorbol 12-myristate 13-acetate (PMA) was abrogated by a PKC-β-specific inhibitor (LY333531). In contrast, the LTR-mediated synergy induced by Ctx and TNFα was not affected by LY333531. The synergy induced by Ctx and TNFα was also abrogated by mutation of the cAMP-responsive downstream sequence elements (DSE) in the 5′ untranslated leader region, whereas the DSE mutations did not affect the synergy induced by Ctx and PMA. These distinctions indicate that Ctx cooperates differently with TNFα and PMA to activate the HIV-1 LTR. Ctx and PMA synergistically activated AP-1- and NF-κB-dependent transcription, even though no cooperative binding of AP-1 or NF-κB was observed in gel shift assays. An extensive mutational analysis of the HIV-1 LTR that included the NF-κB and AP-1 binding sites revealed no distinctcis-acting element or region within the HIV-1 LTR that was required for the transcriptional synergy. Ctx and PMA also synergistically interact to activate the HTLV-1 LTR. These results indicate that the transcriptional synergy elicited by Ctx and PMA targets multiple functional elements and promoters, requires a cooperative interaction between the PKA and PKC-β pathways, and differs mechanistically from the transcriptional synergy induced by Ctx and TNFα.