The immature plasmacytoid dendritic cell (PDC) is identical with the principal type I IFN-producing cell upon viral infection. Oligodeoxynucleotides which contain unmethylated CpG motifs (CpG ODN) are recognized by the vertebrate immune system. Previously, we described CpG ODN that strongly activate human B cells and human blood dendritic cells. Here we describe distinct CpG-containing oligonucleotide sequences which, in contrast to previously described CpG ODN, induced high amounts of IFN-alpha and IFN-beta in peripheral blood mononuclear cells (PBMC). Intracellular staining for IFN-alpha revealed that within PBMC CpG ODN-induced IFN-alpha is produced exclusively by PDC. Unlike IFN-alpha, TNF-alpha is up-regulated in PDC by all CpG ODN tested. Purified PDC responded to CpG ODN, demonstrating direct activation of PDC by CpG ODN. The most active sequence induced the production of up to 5 pg IFN-alpha per single PDC, resulting in more than 400 ng/ml IFN-alpha in the supernatant of PBMC enriched for PDC. The potency of CpG ODN to stimulate IFN-alpha correlated with their ability to stimulate NK cell lytic activity, while purified NK cells did not respond to CpG ODN. IFNgamma production in PBMC was dependent on CpG ODN-induced IFN-alpha/beta as demonstrated by IFN-alpha/beta blocking antibodies. IFN-alpha-inducing CpG ODN strongly supported IFN-gamma production of TCR-triggered CD4 T cells but were less active than other CpG ODN in stimulating B cells. In conclusion our results demonstrate that particular CpG ODN sequences exist which, due to high IFN-alpha/beta induction in PDC, induce a set of immune responses typical for viral infection.