Neonates are more susceptible than adults to viral and bacterial diseases. We hypothesized that plasmacytoid dendritic cells, the cells that provide large amounts of IFN-alpha in response to Toll-like receptor 9 (TLR9) agonists, are defective in neonates. To assess the intrinsic functionality of plasmacytoid dendritic cells from neonates we compared IFN-alpha production by plasmacytoid dendritic cells derived from neonates versus adults in both whole blood and in purified plasmacytoid dendritic cells. TLR9-stimulation of whole blood from adults and neonates resulted in comparable amounts of IFN-alpha production. However, we observed small but significant differences in IFN-alpha production from purified CD123+ plasmacytoid dendritic cells from neonates after stimulation with the TLR9 ligand CpG-DNA. Furthermore, we assessed surface expression of co-stimulatory molecules on plasmacytoid dendritic cells after stimulation. While purified CD123+ plasmacytoid dendritic cells from adults up-regulated co-stimulatory molecules CD80 and CD86 with IL-3 alone those from neonates required the addition of CpG-DNA to reach adult levels. Therefore, the intrinsic deficiencies of neonatal plasmacytoid dendritic cells can be mitigated by TLR9 agonists. These results are consistent with the observation that vaccines that effect strong adjuvant activity on dendritic cells can induce protective responses in neonates.