CpG oligodeoxynucleotides (ODNs) signal through TLR9 to induce type I IFN (IFN-alphabeta) in dendritic cells (DCs). CpG-A ODNs are more efficacious than CpG-B ODNs for induction of IFN-alphabeta. Because IFN-alphabeta may contribute to autoimmunity, it is important to identify mechanisms to inhibit induction of IFN-alphabeta. In our studies, CpG-B ODN inhibited induction of IFN-alphabeta by CpG-A ODN, whereas induction of TNF-alpha and IL-12p40 by CpG-A ODN was not affected. CpG-B inhibition of IFN-alphabeta was observed in FLT3 ligand-induced murine DCs, purified murine myeloid DCs, plasmacytoid DCs, and human PBMCs. CpG-B ODN inhibited induction of IFN-alphabeta by agonists of multiple receptors, including MyD88-dependent TLRs (CpG-A ODN signaling via TLR9, or R837 or Sendai virus signaling via TLR7) and MyD88-independent receptors (polyinosinic:polycytidylic acid signaling via TLR3 or ds break-DNA signaling via a cytosolic pathway). CpG-B ODN did not inhibit the IFN-alphabeta positive feedback loop second-wave IFN-alphabeta, because IFN-alphabeta-induced expression of IFN-alphabeta was unaffected, and CpG-B inhibition of IFN-alphabeta was manifested in IFN-alphabetaR(-/-) DCs, which lack the positive feedback mechanism. Rather, CpG-B ODN inhibited early TLR-induced first wave IFN-alpha4 and IFN-beta. Chromatin immunoprecipitation revealed that association of IFN regulatory factor 1 with the IFN-alpha4 and IFN-beta promoters was induced by CpG-A ODN but not CpG-B ODN. Moreover, CpG-A-induced association of IFN regulatory factor 1 with these promoters was inhibited by CpG-B ODN. Our studies demonstrate a novel mechanism of transcriptional regulation of first-wave IFN-alphabeta that selectively inhibits induction of IFN-alphabeta downstream of multiple receptors and may provide targets for future therapeutic inhibition of IFN-alphabeta expression in vivo.