Human TLR9 recognizes bacterial DNA via species-specific CpG motifs. The study shows that human TLR9 expression in immune cells correlates with responsiveness to CpG-DNA. Expressing TLR9 in nonresponder cells confers immunostimulatory responses to CpG-DNA. Both human and murine TLR9 confer responsiveness in a CD14- and MD2-independent manner, but require species-specific CpG motifs for signaling via MyD88. The optimal CpG motif for hTLR9 is GTCGTT, while for mTLR9 it is GACGTT. These findings suggest that hTLR9 directly engages immunostimulatory CpG-DNA in a species-specific manner. The immune system uses pattern recognition receptors to detect pathogens. TLRs are transmembrane proteins that recognize pathogen-associated molecular patterns. TLRs initiate signaling via MyD88, IRAK, and TRAF6, leading to NF-κB and MAPK activation. TLR2 and TLR4 transduce signals via their TIR domain. Bacterial DNA induces inflammatory responses due to high unmethylated CG dinucleotides. Synthetic CpG-DNA mimics bacterial DNA's immunostimulatory effects. TLR9-deficient mice are nonresponsive to CpG-DNA, suggesting TLR9 is a CpG-DNA receptor. DNA-PKcs-deficient mice are also nonresponsive to CpG-DNA, indicating DNA-PKcs may recognize CpG motifs. CpG-DNA acts as a Th1-biasing adjuvant in mouse models. CpG-DNA activates human cells like dendritic cells and B cells. The optimal CpG motif for human cells differs from that for mice. Expression of hTLR9 correlates with CpG-DNA responsiveness in human cells. Transfection of hTLR9 or mTLR9 into nonresponder cells reconstitutes MyD88-dependent CpG-DNA responses. Transfected hTLR9 and mTLR9 require distinct CpG motifs for signaling, indicating species-specific engagement of CpG-DNA. The study also shows that TLR9 is MyD88-dependent for CpG-DNA signaling. TLR9 confers species-specific CpG motif signaling, with hTLR9 recognizing GTCGTT and mTLR9 recognizing GACGTT. The study confirms that TLR9 directly interacts with CpG-DNA. The TLR9 complementation system allows analysis of species-specific CpG motifs. The human optimal CpG motif differs from the murine one. TLR9 is essential for CpG-DNA signaling, as shown by genetic complementation experiments. TLR9 is independent of MD2 and CD14, unlike TLR4. TLR9 directly engages CpG-DNA, as shown by its DNA-binding motif. The study highlights the roleHuman TLR9 recognizes bacterial DNA via species-specific CpG motifs. The study shows that human TLR9 expression in immune cells correlates with responsiveness to CpG-DNA. Expressing TLR9 in nonresponder cells confers immunostimulatory responses to CpG-DNA. Both human and murine TLR9 confer responsiveness in a CD14- and MD2-independent manner, but require species-specific CpG motifs for signaling via MyD88. The optimal CpG motif for hTLR9 is GTCGTT, while for mTLR9 it is GACGTT. These findings suggest that hTLR9 directly engages immunostimulatory CpG-DNA in a species-specific manner. The immune system uses pattern recognition receptors to detect pathogens. TLRs are transmembrane proteins that recognize pathogen-associated molecular patterns. TLRs initiate signaling via MyD88, IRAK, and TRAF6, leading to NF-κB and MAPK activation. TLR2 and TLR4 transduce signals via their TIR domain. Bacterial DNA induces inflammatory responses due to high unmethylated CG dinucleotides. Synthetic CpG-DNA mimics bacterial DNA's immunostimulatory effects. TLR9-deficient mice are nonresponsive to CpG-DNA, suggesting TLR9 is a CpG-DNA receptor. DNA-PKcs-deficient mice are also nonresponsive to CpG-DNA, indicating DNA-PKcs may recognize CpG motifs. CpG-DNA acts as a Th1-biasing adjuvant in mouse models. CpG-DNA activates human cells like dendritic cells and B cells. The optimal CpG motif for human cells differs from that for mice. Expression of hTLR9 correlates with CpG-DNA responsiveness in human cells. Transfection of hTLR9 or mTLR9 into nonresponder cells reconstitutes MyD88-dependent CpG-DNA responses. Transfected hTLR9 and mTLR9 require distinct CpG motifs for signaling, indicating species-specific engagement of CpG-DNA. The study also shows that TLR9 is MyD88-dependent for CpG-DNA signaling. TLR9 confers species-specific CpG motif signaling, with hTLR9 recognizing GTCGTT and mTLR9 recognizing GACGTT. The study confirms that TLR9 directly interacts with CpG-DNA. The TLR9 complementation system allows analysis of species-specific CpG motifs. The human optimal CpG motif differs from the murine one. TLR9 is essential for CpG-DNA signaling, as shown by genetic complementation experiments. TLR9 is independent of MD2 and CD14, unlike TLR4. TLR9 directly engages CpG-DNA, as shown by its DNA-binding motif. The study highlights the role