Combating tuberculosis requires a detailed understanding of how mycobacterial effectors modulate the host immune response. The role of the multigene PE family of proteins unique to mycobacteria in the pathogenesis of tuberculosis is still poorly understood, although certain PE_PGRS genes have been linked to virulence. Tumor necrosis factor-alpha (TNF-alpha) is essential for successfully combating tuberculosis. In this study we provide evidence that PE_PGRS33, a surface exposed protein, elicits TNF-alpha release from macrophages in a TLR2 (Toll-like receptor 2)-dependent manner. ASK1 (apoptosis signal-regulating kinase 1) is activated downstream of TLR2. ASK1 activates the MAPKs p38 and JNK. PE_PGRS33-induced signaling leads to enhanced expression of TNF-alpha and TNF receptor I (TNFRI) genes. Mycobacterium smegmatis expressing PE_ PGRS33 elicits the same effects as purified PE_PGRS33. TNF-alpha release occurs even when internalization of the bacteria is blocked by cytochalasin D, suggesting that interaction of PE_ PGRS33 with TLR2 is sufficient to trigger the effects described. Release of TNF-alpha plays the determining role in triggering apoptosis in macrophages challenged with PE_PGRS33. The death receptor-dependent signals are amplified through classical caspase 8-dependent mitochondrial release of cytochrome c, leading to the activation of caspases 9 and 3. An important aspect of our findings is that deletions within the PGRS domain (simulating those occurring in clinical strains) attenuate the TNF-alpha-inducing ability of PE_PGRS33. These results provide the first evidence that variations in the polymorphic repeats of the PGRS domain modulate the innate immune response.