Immunostimulatory sequences (ISS) are short oligonucleotides containing unmethylated cytosine-phosphate-guanine (CpG) dinucleotides that stimulate innate immune responses through Toll-like receptor-9 on B cells and plasmacytoid dendritic cell (PDC) precursors. The anti-inflammatory cytokine interleukin (IL)-10 is predicted to be a potent inhibitor of many of the activities described for ISS, and this may impact the use of ISS in disease states characterized by elevated IL-10. As the activities of ISS on PDCs are central to many clinical applications of ISS, we have studied the effects of IL-10 on PDC stimulation by 3 distinct classes of ISS. IL-10 inhibited cytokine production and survival of ISS-activated PDCs; however, IL-12 induction was much more sensitive to inhibition than interferon (IFN)-alpha induction. Within the PDC population are cells that respond to ISS by producing either IL-12 or IFN-alpha but not both cytokines. IL-12-producing PDCs require costimulation through CD40 and appear more mature than IFN-alpha-producing PDCs. The 3 distinct classes of ISS differed with respect to induction of PDC maturation and T-cell priming capacity. IL-10 regulated PDC activation but did not inhibit the subsequent T-cell-priming ability of PDCs already activated by ISS.