CpG-oligonucleotides (CpG-ODN) have been shown to exert strong immuno-stimulatory effects through activation of Toll-like receptor 9 (TLR-9). However, TLR-9 triggering takes place in endosomal compartments and thus CpG-ODN have to be taken up prior to signal transduction. We here report that 3'-poly-guanosine strings can improve cellular internalisation of phosphodiester but not of phosphorothioate CpG-ODN. Improved cellular uptake correlated with enhanced IL-6 secretion and proliferation of PBMC. Also, TLR-9 transfected HEK293 cells were activated more efficiently by poly-guanosine modified CpG-ODN. The results indicate that the synthesis of stimulatory CpG-ODN based on a phosphodiester backbone is feasible via such poly-guanosine substitutions. In addition we observed that phosphorothioate ODN were able to exert immunostimulatory effects independent of the presence of CpG motifs.