Polyinosinic:polycytidylic acid (poly IC), a double-stranded RNA, is an effective adjuvant in vivo. IFN-s (also termed IL-28/29) are potent immunomodulatory and antiviral cytokines. We demonstrate that poly IC injection in vivo induces large amounts of IFN-, which depended on hematopoietic cells and the presence of TLR3 (Toll-like receptor 3), IRF3 (IFN regulatory factor 3), IRF7, IFN-I receptor, Fms-related tyrosine kinase 3 ligand (FL), and IRF8 but not on MyD88 (myeloid differentiation factor 88), Rig-like helicases, or lymphocytes. Upon poly IC injection in vivo, the IFN- production by splenocytes segregated with cells phenotypically resembling CD8⁺ conventional dendritic cells (DCs [cDCs]). In vitro experiments revealed that CD8⁺ cDCs were the major producers of IFN- in response to poly IC, whereas both CD8⁺ cDCs and plasmacytoid DCs produced large amounts of IFN- in response to HSV-1 or parapoxvirus. The nature of the stimulus and the cytokine milieu determined whether CD8⁺ cDCs produced IFN- or IL-12p70. Human DCs expressing BDCA3 (CD141), which is considered to be the human counterpart of murine CD8⁺ DCs, also produced large amounts of IFN- upon poly IC stimulation. Thus, IFN- production in response to poly IC is a novel function of mouse CD8⁺ cDCs and their human equivalents.