Toll-like receptor 7 (TLR7) and TLR8 are considered to recognize single-strand RNA (ssRNA) from viruses in endolysosomes and induce antiviral immune response. In addition, these receptors respond to synthetic small molecules, such as R848 and CL075. However, it remains unclear how those two different types of ligands can activate both TLR7 and TLR8. In the crystal structure of human TLR8 (huTLR8) complexed with ssRNA, there were two ligand-binding sites; the first site, a binding site for R848, bound a uridine and the second site bound an oligoribonucleotide (ORN). In reporter assay using HEK293T cells, uridine and ORNs synergistically activated huTLR8, suggesting that huTLR8 recognizes degradation products of ssRNA. We also show that TLR7 recognizes guanosine (G) in the presence of ORN. G and ORN synergistically activated TLR7 in plasmacytoid DCs and induced IFN-a production. By isothermal titration calorimetry experiment, specific binding of G to TLR7/ORN complex was detected and the affinity of G to TLR7/ORN complex was determined to be Kd=1.5μM. These results strongly suggest that TLR7 also recognizes degradation products of ssRNA, guanosine and ORN, but not ssRNA itself. In conclusion, endolysosomal nucleosides play a decisive role in TLR7 and TLR8 activation by ssRNA.