Opportunistic infections are common in HIV-infected patients; they activate HIV replication and contribute to disease progression. In the present study we examined the role of Toll-like receptor 2 (TLR2) and TLR9 in HIV-long terminal repeat (HIV-LTR) trans-activation and assessed whether TLR4 synergized with TLR2 or TLR9 to induce HIV replication. Soluble Mycobacterium tuberculosis factor (STF) and phenol-soluble modulin from Staphylococcus epidermidis induced HIV-LTR trans-activation in human microvessel endothelial cells cotransfected with TLR2 cDNA. Stimulation of ex vivo spleen cells from HIV-1 transgenic mice with TLR4, TLR2, and TLR9 ligands (LPS, STF, and CpG DNA, respectively) induced p24 Ag production in a dose-dependent manner. Costimulation of HIV-1 transgenic mice spleen cells with LPS and STF or CpG DNA induced TNF-alpha and IFN-gamma production in a synergistic manner and p24 production in an additive fashion. In the THP-1 human monocytic cell line stably expressing the HIV-LTR-luciferase construct, LPS and STF also induced HIV-LTR trans-activation in an additive manner. This is the first time that TLR2 and TLR9 and costimulation of TLRs have been shown to induce HIV replication. Together these results underscore the importance of TLRs in bacterial Ag- and CpG DNA-induced HIV-LTR trans-activation and HIV replication. These observations may be important in understanding the role of the innate immune system and the molecular mechanisms involved in the increased HIV replication and HIV disease progression associated with multiple opportunistic infections.