DNA from bacteria has stimulatory effects on mammalian immune cells, which depend on the presence of unmethylated CpG dinucleotides in the bacterial DNA. In contrast, mammalian DNA has a low frequency of CpG dinucleotides, and these are mostly methylated; therefore, mammalian DNA does not have immuno-stimulatory activity. CpG DNA induces a strong T-helper-1-like inflammatory response. Accumulating evidence has revealed the therapeutic potential of CpG DNA as adjuvants for vaccination strategies for cancer, allergy and infectious diseases. Despite its promising clinical use, the molecular mechanism by which CpG DNA activates immune cells remains unclear. Here we show that cellular response to CpG DNA is mediated by a Toll-like receptor, TLR9. TLR9-deficient (TLR9-/-) mice did not show any response to CpG DNA, including proliferation of splenocytes, inflammatory cytokine production from macrophages and maturation of dendritic cells. TLR9-/- mice showed resistance to the lethal effect of CpG DNA without any elevation of serum pro-inflammatory cytokine levels. The in vivo CpG-DNA-mediated T-helper type-1 response was also abolished in TLR9-/- mice. Thus, vertebrate immune systems appear to have evolved a specific Toll-like receptor that distinguishes bacterial DNA from self-DNA.