Recent reports have identified two major classes of CpG motif-containing oligodeoxynucleotide immunostimulatory sequences (ISS): uniformly modified phosphorothioate (PS) oligodeoxyribonucleotides (ODNs), which initiate B cell functions but poorly activate dendritic cells (DCs) to make interferon (IFN)-α, and chimeric PS/phosphodiester (PO) ODNs containing runs of six contiguous guanosines, which induce very high levels of plasmacytoid DC (PDC)-derived IFN-α but poorly stimulate B cells. We have generated the first reported ISS, C274, which exhibits very potent effects on all human immune cells known to recognize ISS. C274 is a potent inducer of IFN-γ/IFN-α from peripheral blood mononuclear cells and exhibits accelerated kinetics of activity compared with standard ISS. This ODN also effectively stimulates B cells to proliferate, secrete cytokines, and express costimulatory antigens. In addition, C274 specifically activates PDCs to undergo maturation and secrete cytokines, including very high levels of IFN-α. Sequence variation studies based on C274 were used to identify the general motif requirements for this novel and distinct class of ISS. In contrast, chimeric PO/PS CpG-containing ODNs with polyguanosine sequences exert a differential pattern of ISS activity compared with C274, perhaps in part as a result of their greatly different structural nature. This pattern is composed of high IFN-α/IFN-γ induction and low DC maturation in the absence of B cell stimulation. In conclusion, we have generated a novel class of ISS that transcends the limitations ascribed to classes described previously in that it provides excellent stimulation of B cells and simultaneously activates PDCs to differentiate and secrete large amounts of type I IFN.