Synthetic immune response modifiers (IRM) such as imidazoquinolines can selectively activate human TLR7 or TLR8. Although these endosomal TLRs are close relatives, TLR7-deficient mice are unresponsive to TLR8 agonist IRMs. Similarly, natural ssRNA cannot activate murine TLR8, leading to the belief that murine TLR8 is nonfunctional. In this study, we transfected HEK293 cells with murine TLR8 and NF-κB reporter constructs and stimulated them with combinations of IRM and oligodeoxynucleotides (ODNs). When stimulated with TLR7 or TLR8 agonists alone, no NF-κB response was observed. However, a combination of polyT ODN plus the TLR8 agonist activated NF-κB, whereas polyT ODN plus the TLR7 agonist did not activate. Primary mouse cells responded to the IRM/polyT ODN by secreting TNF. Cells from TLR7−/− and TLR9−/− mice responded to the IRM/polyT ODN combination, whereas MyD88−/− cells did not respond. In conclusion, this study demonstrates for the first time that mouse TLR8 is functional.