In the course of the development of acquired immunodeficiency syndrome (AIDS), bacterial infection causes deleterious effects on the progression of the disease; bacterial LPS in the circulation activate immune cells, resulting in the acceleration of HIV replication. However, the precise HIV activation mechanisms in infected hosts remain largely unknown. Previously, we generated transgenic (Tg) mice carrying the HIV type I (HIV-1) genome and showed that LPS induces the activation of HIV-1 in splenocytes through the induction of tumor necrosis factor (TNF) and IL-1, although similarly induced IFN-gamma and IL-6 are not involved. In this study, we analyzed the mechanisms of HIV-1 activation in macrophages using these HIV-1 Tg mice, because macrophages are one of the major reservoirs of HIV-1. In contrast to splenocytes, direct Toll-like receptor (TLR) 4 signaling rather than TLR-induced pro-inflammatory cytokines was responsible for the LPS-induced activation of HIV-1 in macrophages, because the time course of HIV-1 activation was earlier than that observed in splenocytes and TNF neutralization did not inhibit the activation. p38 mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-kappaB) activation, but neither extracellular signal-regulated kinase nor c-Jun N-terminal kinase activation, were required for the activation, because only inhibitors for p38 MAPK and NF-kappaB suppressed activation of HIV-1. Furthermore, we showed that myeloid differentiation primary response gene (MyD) 88, rather than Toll/IL-1R domain-containing adaptor inducing IFN-beta (TRIF), was required as an adaptor molecule for this activation using Myd88(-/-) mice and Dynasore, a specific inhibitor for TRIF, and small interfering RNAs specific for Myd88 and Trif. These observations suggest that suppression of these molecules, which are involved in the TLR4-MyD88 pathway and the downstream p38 MAPK and NF-kappaB pathways, should be beneficial to prevent development of AIDS in HIV-1-infected people.