Human plasmacytoid or CD4(+)CD11c(-) type 2 dendritic cell precursors (PDC) were identified as natural type I interferon (IFN)-producing cells in response to viral and bacterial infection. They represent effector cells of innate immunity and link it to the distinct adaptive immunity by differentiating into mature DC. It has been reported that oligodeoxyribonucleotides containing unmethylated CpG motifs (CpG DNA) stimulate PDC to produce IFN-alpha, but the molecular mechanisms involved remain unknown. We found that CpG-DNA-induced IFN-alpha production in PDC was completely impaired by the inhibitor of the p38 mitogen-activated protein kinase (MAPK) pathway. Expression of IFN regulatory factor (IRF)-7 was enhanced by CpG-DNA treatment, which was preceded by the phosphorylation of signal transducer and activator of transcription (STAT)1 on Tyr-701, as well as its enhanced phosphorylation on Ser-727. All of these events were also suppressed by the p38 MAPK inhibitor. STAT1, STAT2, and IRF-9, components of IFN-stimulated gene factor 3 (ISGF3), were recognized in the nuclear fraction of CpG-DNA-treated cells. Neither anti-IFN-alpha/beta antibodies (Ab) nor anti-IFNAR Ab suppressed STAT1 phosphorylation, enhancement of IRF-7 expression, or IFN-alpha production in the early phase of the culture. These results suggest that CpG DNA induces p38 MAPK-dependent phosphorylation of STAT1 in a manner independent of IFN-alpha/beta, which may cause ISGF3 formation to increase the transcription of the IRF-7 gene, thereby leading to IFN-alpha production in human PDC.