Foreign nucleic acids, the signature of invading viruses and certain bacteria, are sensed intracellularly. The nucleic acid-specific Toll-like receptors (TLRs) detect and signal within endolysosomal compartments, triggering the induction of cytokines essential for the innate immune response. These cytokines include proinflammatory molecules produced mainly by macrophages and conventional dendritic cells, as well as type I interferons, which are produced in great quantities by plasmacytoid dendritic cells. The cellular and molecular pathways by which nucleic acids and TLRs meet within the endosome assure host protection yet also place the host at risk for the development of autoimmunity. Here, we review the latest findings on the intracellular TLRs, with special emphasis on ligand uptake, receptor trafficking, signaling, and regulation.