Among the bacterial products known to activate the innate immune ‘1system is bacterial DNA. This activity resides within the nonmethylated CpG motifs of the DNA and is recapitulated using appropriate synthetic CpG containing oligodeoxynucleotides (CpG-ODN). TLR9-deficient mice were shown to exhibit a nonresponsive phenotype-to-bacterial DNA and CpG-ODN. Here, we describe a model system to further characterize CpG-ODN and TLR9 interactions using ectopically expressed TLR9 in HEK293 cells. Expression of TLR9 confers cellular responsiveness to CpG-ODN but not to the other bacterial products. Previous studies identified species-specific CpG-containing sequences; here, we show that expression of murine TLR9 favors responses to CpG-ODN motifs specific to mouse cells, and expression of human TLR9 favors CpG-ODN known to preferentially activate human cells. Response patterns to various CpG-ODN motifs were parallel when cells containing an ectopically expressed TLR9 and endogenous receptor were compared. Here, we also show that TLR9 acts at the cell surface and engages an intracellular signaling pathway that includes MyD88, IRAK, and TRAF6.