A recent report demonstrated that free human T-cell leukemia virus 1 (HTLV-1) could infect plasmacytoid dendritic cells (pDCs). The major role of pDCs is to secrete massive levels of interferon-alpha (IFN-alpha) upon virus exposure; however, the induction of IFN-alpha by HTLV-1 remains unknown. We demonstrate here that cell-free HTLV-1 generated a pDC innate immune response by producing massive levels of IFN-alpha that were inhibited by anti-HTLV-1 antibodies. HTLV-1 induced costimulatory molecules and rapid expression of the apoptotic ligand tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Furthermore, HTLV-1 stimulated pDC-induced apoptosis of CD4(+) T cells expressing DR5, transforming pDCs into IFN-producing killer pDCs. We also observed that an endosomal acidification inhibitor and a Toll-like receptor-7 (TLR7)-specific blocker drastically inhibited pDC response to HTLV-1. Three-dimensional microscopy analysis revealed that unstimulated pDCs were "dormant" IFN-producing killer pDCs with high levels of intracellular TRAIL that could be rapidly mobilized to the surface in response to TLR7 activation. Inhibition of viral degradation in endosomes by chloroquine maintained viral integrity, allowing virus detection by 3-dimensional microscopy. We demonstrate that pDCs respond to cell-free HTLV-1 by producing high levels of IFN-alpha and by mobilizing TRAIL on cell surface after TLR7 triggering. This is the first demonstration of an innate immune response induced by free HTLV-1.