TLR2 activation plays a crucial role in Neisseria gonorrheae-mediated enhancement of HIV infection of resting CD4(+) T cells. We examined signaling pathways involved in the HIV enhancing effect of TLR2. TLR2 but not IL-2 signals promoted HIV nuclear import; however, both signals were required for the maximal enhancing effect. Although TLR2 signaling could not activate T cells, it increased IL-2-induced T cell activation. Cyclosporin A and IkBα inhibitor blocked TLR2-mediated enhancement of HIV infection/nuclear import. PI3K inhibitor blocked HIV infection/nuclear import and T cell activation and exerted a moderate inhibitory effect on cell cycle progression in CD4(+) T cells activated by TLR2/IL-2. Blockade of p38 signaling suppressed TLR2-mediated enhancement of HIV nuclear import/infection. However, the p38 inhibitor did not have a significant effect on T cell activation or TCR/CD3-mediated enhancement of HIV infection/nuclear import. The cell cycle arresting reagent aphidicolin blocked TLR2- and TCR/CD3-induced HIV infection/nuclear import. Finally, cyclosporin A and IκBα and PI3K inhibitors but not the p38 inhibitor blocked TLR2-mediated IκBα phosphorylation. Our results suggest that TLR2 activation enhances HIV infection/nuclear import in resting CD4(+) T cells through both T cell activation-dependent and -independent mechanisms.