Over the past decade we have learned much about nucleic acid recognition by the innate immune system and in particular by Toll-like receptors (TLRs). These receptors localize to endosomal compartments where they are poised to recognize microbial nucleic acids. Multiple regulatory mechanisms function to limit responses to self DNA or RNA, and breakdowns in these mechanisms can contribute to autoimmune or inflammatory disorders. In this review we discuss our current understanding of the cell biology of TLRs involved in nucleic acid recognition and how localization and trafficking of these receptors regulates their function.