Toll-like receptor-2 (TLR2) mediates mycobacteria-induced proinflammatory signaling in macrophages. This study shows that TLR2 is essential for the induction of tumor necrosis factor-alpha (TNF-α) in macrophages by Mycobacterium tuberculosis. Expression of a dominant negative form of MyD88, a signaling component required for toll-like receptor signaling, blocks TNF-α production induced by M. tuberculosis. TLR2 is identified as the specific toll-like receptor required for this induction. TLR2-dependent signaling mediates responses to mycobacterial cell wall fractions enriched for lipoarabinomannan, mycolylarabinogalactan-peptidoglycan complex, or M. tuberculosis total lipids. These data suggest that TLR2 is essential for the induction of a protective immune response to mycobacteria. TLR2 is required for the detection of mycobacteria by macrophages and clearly demonstrates functional specificity of toll-like receptors for particular ligands. TLR2 is also involved in the recognition of Gram-positive bacteria, lipoteichoic acid, peptidoglycan, and yeast cell walls. TLR2 may oligomerize with other toll-like receptors, thus accounting for its apparently broad ligand specificity. TLR2-dependent signals are likely to participate in innate inflammatory responses to mycobacteria in a manner analogous to the role of TLR4 in host response to Gram-negative bacterial infections. This study was supported by grants from the National Institutes of Health.Toll-like receptor-2 (TLR2) mediates mycobacteria-induced proinflammatory signaling in macrophages. This study shows that TLR2 is essential for the induction of tumor necrosis factor-alpha (TNF-α) in macrophages by Mycobacterium tuberculosis. Expression of a dominant negative form of MyD88, a signaling component required for toll-like receptor signaling, blocks TNF-α production induced by M. tuberculosis. TLR2 is identified as the specific toll-like receptor required for this induction. TLR2-dependent signaling mediates responses to mycobacterial cell wall fractions enriched for lipoarabinomannan, mycolylarabinogalactan-peptidoglycan complex, or M. tuberculosis total lipids. These data suggest that TLR2 is essential for the induction of a protective immune response to mycobacteria. TLR2 is required for the detection of mycobacteria by macrophages and clearly demonstrates functional specificity of toll-like receptors for particular ligands. TLR2 is also involved in the recognition of Gram-positive bacteria, lipoteichoic acid, peptidoglycan, and yeast cell walls. TLR2 may oligomerize with other toll-like receptors, thus accounting for its apparently broad ligand specificity. TLR2-dependent signals are likely to participate in innate inflammatory responses to mycobacteria in a manner analogous to the role of TLR4 in host response to Gram-negative bacterial infections. This study was supported by grants from the National Institutes of Health.