Toll-like receptors (TLRs) sense infection by detecting molecular structures of microbial origin. TLR3, TLR7 and TLR9 recognize nucleic acids and are localized to intracellular compartments where they normally respond to viral nucleic acids. The purpose for this intracellular localization, however, is not clear. Here we describe a chimeric TLR9 receptor that localized to the cell surface and responded normally to synthetic TLR9 ligands but not to viral nucleic acids. However, the 'relocated' chimeric TLR9 receptor was able to recognize self DNA, which does not stimulate wild-type TLR9. These data demonstrated that intracellular localization of TLR9 was not required for ligand recognition. Instead, localization of the nucleic acid-sensing TLRs is critical in discriminating between self and nonself nucleic acid.