The mammalian innate immune system retains fromDrosophila a family of homologous Toll-like receptors (TLRs) that mediate responses to microbial ligands. Here, we show that TLR2 activation leads to killing of intracellular Mycobacterium tuberculosis in both mouse and human macrophages, through distinct mechanisms. In mouse macrophages, bacterial lipoprotein activation of TLR2 leads to a nitric oxide–dependent killing of intracellular tubercle bacilli, but in human monocytes and alveolar macrophages, this pathway was nitric oxide–independent. Thus, mammalian TLRs respond (asDrosophila Toll receptors do) to microbial ligands and also have the ability to activate antimicrobial effector pathways at the site of infection.