TLR9 is critical in parasite recognition and host resistance to experimental infection with Trypanosoma cruzi. However, no information is available regarding nucleotide sequences and cellular events involved on T. cruzi recognition by TLR9. In silico wide analysis associated with in vitro screening of synthetic oligonucleotides demonstrates that the retrotransposon VIPER elements and mucin-like glycoprotein (TcMUC) genes in the T. cruzi genome are highly enriched for CpG motifs that are immunostimulatory for mouse and human TLR9, respectively. Importantly, infection with T. cruzi triggers high levels of luciferase activity under NF-kappaB-dependent transcription in HEK cells cotransfected with human TLR9, but not in control (cotransfected with human MD2/TLR4) HEK cells. Further, we observed translocation of TLR9 to the lysosomes during invasion/uptake of T. cruzi parasites by dendritic cells. Consistently, potent proinflammatory activity was observed when highly unmethylated T. cruzi genomic DNA was delivered to the endo-lysosomal compartment of host cells expressing TLR9. Thus, together our results indicate that the unmethylated CpG motifs found in the T. cruzi genome are likely to be main parasite targets and probably become available to TLR9 when parasites are destroyed in the lysosome-fused vacuoles during parasite invasion/uptake by phagocytes.