Experiments were performed to determine the contribution of TLR9 to the generation of protective immunity against the intracellular respiratory bacterial pathogen Legionella pneumophila. In initial studies, we found that the intratracheal (i.t.) administration of L. pneumophila to mice deficient in TLR9 (TLR9(-/-)) resulted in significantly increased mortality, which was associated with an approximately 10-fold increase in the number of lung CFU compared to that of wild-type BALB/c mice. Intrapulmonary bacterial challenge in TLR9(-/-) mice resulted in the reduced accumulation of myeloid dendritic cells (DC) and activated CD4(+) T cells. Lung macrophages isolated from Legionella-infected TLR9(-/-) mice displayed the impaired internalization of bacteria and evidence of alternative rather than classical activation, as manifested by the markedly reduced expression of nitric oxide and type 1 cytokines, whereas the expression of Fizz-1 and arginase-1 was enhanced. The adoptive transfer of bone marrow-derived DC from syngeneic wild-type, but not TLR9(-/-), mice administered i.t. reconstituted anti-legionella immunity and restored the macrophage phenotype in TLR9(-/-) mice. Finally, the i.t., but not intraperitoneal, administration of the TLR9 agonist molecule CpG oligodeoxynucleotide stimulated protective immunity in Legionella-infected mice. In total, our findings indicate that TLR9 is required for effective innate immune responses against the intracellular bacterial pathogen L. pneumophila, and approaches to maximize TLR9-mediated responses may serve as a means to augment antibacterial immunity in pneumonia.