Plasmacytoid dendritic cells (pDCs) play an important role in innate and adaptive immunity and were shown to be identical to previously described natural interferon (IFN)-α-producing cells. Here, we describe two functionally distinct pDC subpopulations that are characterized by the differential expression of stem cell antigen-1 (Sca-1; Ly-6A/E). Sca-1− pDCs are mainly found in the BM, appear first during development, show a higher proliferative activity, and represent the more precursor phenotype. Sca-1+ pDCs are mostly located in secondary lymphoid organs and represent a later developmental stage. Sca-1− pDCs give rise to an Sca-1+ subset upon activation or in response to endogenous type I IFN. Interestingly, in contrast to Sca-1− pDCs, Sca-1+ pDCs are defective in IFN-α production upon endosomal TLR9 stimulation, whereas lysosomal signaling via TLR9 is functional in both subsets. Gene expression analysis revealed that osteopontin is strongly upregulated in Sca-1− pDCs. These data provide evidence for the molecular basis of the observed functional heterogeneity, as the intracellular isoform of osteopontin couples TLR9 signaling to IFN-α expression. Taken together, our results indicate that Sca-1− pDCs are an early developmental stage of pDCs with distinct innate functions representing the true murine natural IFN-α-producing cells.