While initial studies of Toll-like Receptor (TLR) signaling mainly focused on genetic analysis of signal transduction, recent work has highlighted the importance of understanding the basic cell biology underlying receptor function. Nowhere is this issue more important than in the study of the nucleic acid-sensing TLRs. These receptors face the unique challenge of distinguishing microbial nucleic acids from similar host-derived molecules. The physiological cost of not making this distinction can be readily observed in studies of autoimmunity, a cause of which is often the inappropriate detection of self nucleic acids. In this review, we highlight recent research that has revealed myriad ways in which mammalian cells control the function of nucleic acid-sensing TLRs. A theme is now emerging whereby these receptors are subject to sequential regulatory mechanisms that control protein transport to their sites of signal transduction, as well as their access microbial nucleic acids.