Type 1 (Th1) granulomas can be studied in mice sensitized with mycobacterium antigens followed by challenge of agarose beads covalently coupled to purified protein derivative. TLR9 is known to play a role in the regulation of Th1 responses; thus, we investigated the role of TLR9 in granuloma formation during challenge with mycobacterium antigens and demonstrated that mice deficient in TLR9 had increased granuloma formation, but a dramatically altered cytokine phenotype. Th1 cytokine levels of IFN-gamma and IL-12 in the lungs were decreased in TLR9(-/-) mice when compared to wild-type mice. In contrast, Th2 cytokine levels of IL-4, IL-5, and IL-13 were increased in TLR9(-/-) mice. The migration of CD4(+) T cells in the granuloma was impaired, while the number of F4/80(+) macrophages was increased in TLR9(-/-) mice. Macrophages in the lungs of the TLR9-deficient animals with developing granulomas expressed significantly lower levels of the classically activated macrophage marker, nitric oxide synthase, but higher levels of the alternatively activated macrophage markers such as 'found in inflammatory zone-1' antigen and Arginase-1. These results suggest that TLR9 plays an important role in maintaining the appropriate phenotype in a Th1 granulomatous response.