Natural interferon-producing cells (NIPC), also referred to as immature plasmacytoid dendritic cells (PDC), constitute a small population of leucocytes secreting high levels of type I interferons in response to certain danger signals. Amongst these signals are those from DNA containing unmethylated CpG motifs. The present work demonstrated that the CpG oligonucleotides (CpG-ODN) 2216, D32 and D19 induce high amounts of interferon-α (IFN-α), tumour-necrosis factor-α (TNF-α) and interleukin (IL)-12 in porcine peripheral blood mononuclear cells (PBMCs). Swine workshop cluster 3 (SWC3)1ow CD4high cells, with high IL-3-binding activity, representing NIPC, were the exclusive cytokine-producing cells responding to the CpG-ODN. These cells did not express CD6, CD8 or CD45RA. Importantly, monocyte-derived DC did not respond to CpG-ODN by secretion of IFN-α or TNF-α or by the up-regulation of costimulatory molecule expression. CpG-ODN up-regulated MHC class II and CD80/86 expression on the NIPC, but were unable to promote NIPC survival. Interestingly, certain CpG-ODN, incapable of inducing NIPC to secrete IFN-α or up-regulate MHC class II and CD80/86, did promote NIPC viability. Taken together, the influence of CpG-ODN on porcine NIPC, monocytes and myeloid DCs relates to that observed with their human equivalents. These results represent an important basis for the application of CpG-ODN as adjuvants for the formulation of novel vaccines and demonstrate the importance of the pig as an alternative animal model for this approach.