The HIV-1 LTR is regulated by multiple signaling pathways responsive to T cell activation. In this study, we have examined the contribution of the MAPK, calcineurin-NFAT and TNFalpha-NF-kappaB pathways on induction of chromosomally integrated HIV-1 LTR reporter genes. We find that induction by T-cell receptor (CD3) cross-linking and PMA is completely dependent upon a binding site for RBF-2 (USF1/2-TFII-I), known as RBEIII at -120. The MAPK pathway is essential for induction of the wild type LTR by these treatments, as the MEK inhibitors PD98059 and U0126 block induction by both PMA treatment and CD3 cross-linking. Stimulation of cells with ionomycin on its own has no effect on the integrated LTR, indicating that calcineurin-NFAT is incapable of causing induction in the absence of additional signals, but stimulation with both PMA and ionomycin produces a synergistic response. In contrast, stimulation of NF-kappaB by treatment with TNFalpha causes induction of both the wild type and RBEIII mutant LTRs, an effect that is independent of MAPK signaling. USF1, USF2 and TFII-I from unstimulated cells are capable of binding RBEIII in vitro, and furthermore can be observed on the LTR in vivo by chromatin imunoprecipitation from untreated cells. DNA binding activity of USF1/2 is marginally stimulated by PMA/ ionomycin treatment, and all three factors appear to remain associated with the LTR throughout the course of induction. These results implicate major roles for the MAPK pathway and RBF-2 (USF1/2-TFII-I) in coordinating events necessary for transition of latent integrated HIV-1 to active transcription in response to T cell signaling.