Single-stranded versus multimeric phosphorothioate-modified CpG oligodeoxynucleotides (ODNs) undergo differential endosomal trafficking upon uptake into plasmacytoid dendritic cells (pDCs), correlating with Toll-like receptor-9-dependent pDC maturation/activation (single-stranded B-type CpG ODN) or interferon-alpha (IFN-alpha) induction (multimeric A-type CpG ODN), respectively. As was recently shown, IFN-alpha production, other than by CpG ODNs, can also be induced in a sequence-independent manner by phosphodiester (PD) ODNs multimerized by 3' poly-guanosine (poly-G) tails. We investigate here the type of endosomal trafficking responsible for IFN-alpha induction by natural PD ODN ligands. We show that 3' extension with poly-G tails leads to multimerization of single-stranded PD ODNs and to enhanced cellular uptake into pDCs. While monomeric PD ODNs, which induce CpG-dependent Toll-like receptor-9 stimulation and pDC maturation/activation, localized to late endosomal/lysosomal compartments, the poly-G multimerized PD ODNs, which induce CpG-independent IFN-alpha production, located to vesicles with a distinct, 'early' endosomal phenotype. We conclude that poly-G-mediated multimerization of natural PD ODNs allows for sequence-independent, Toll-like receptor-9-dependent IFN-alpha induction in pDCs by combining three distinct effects: relative protection of sensitive PD ODNs from extracellular and intracellular DNase degradation, enhanced cellular uptake and preferential early endosomal compartmentation.