For cross-presentation immature dendritic cells (DC) require enhanced antigen (Ag) uptake and a maturation signal to prime for MHC class I-restricted CTL responses in vivo. While immunostimulatory CpG-DNA provides, via TLR9, the maturation signal, CpG-DNA linked to Ag augments cellular Ag uptake. In this study we show that CpG-DNA ovalbumin (OVA) conjugates trigger in vivo peptide-specific CTL responses at tenfold lower Ag doses compared to a mixture of CpG-DNA plus OVA. We provide evidence that CpG-DNA-OVA conjugates shift OVA uptake by immature DC from the presumably inefficient fluid phase pinocytosis to efficient DNA receptor-mediated endocytosis. Since the DNA-binding receptor mediating endocytosis lacks any sequence specificity, cellular uptake of OVA conjugated with either stimulatory or non-stimulatory oligonucleotides (ODN) is equally enhanced. As a consequence cross-linking of OVA with either stimulatory or non-stimulatory DNA yields, via enhanced OVA uptake, efficient generation and presentation of the dominant OVA-CTL epitope SIINFEKL. However, only stimulatory CpG-ODN cross-linked to OVA provide the DC maturation signal required to trigger robust primary CTL responses towards the cross-presented MHC class I complexed T cell epitope SIINFEKL. Our studies show that stimulatory CpG-ODN linked to Ag fulfill a dual role: enhancement of Ag uptake yielding efficient Ag cross-presentation by DC and in addition, their activation into professional DC.