The roles of intracellular reactive oxygen species (ROS) and related signalling pathways in mycobacterial infection are largely unknown. Here we show that tuberculin purified protein derivative (PPD)/Toll-like receptor (TLR) 2/ROS signalling through activation of apoptosis-regulating signal kinase (ASK) 1 and p47phox pathways is responsible for the induction of proinflammatory responses during tuberculosis (TB) infection. Tuberculin PPD stimulation resulted in rapid activation of mitogen-activated protein kinases (MAPKs) and an early burst of ROS in monocytes/macrophages in a TLR2-dependent manner. PPD-induced ROS production led to robust activation of ASK1 upstream of p38 MAPK, via TLR2. Interestingly, phosphorylation of the cytosolic NADPH oxidase subunit p47phox and ASK1 activation are mutually dependent on PPD/TLR2-mediated signalling. Furthermore, active pulmonary TB patients showed upregulated ROS generation, as well as enhanced activation of ASK1/p38/p47phox pathways in their primary monocytes compared with healthy controls, which suggests a systemic primed status during TB. Taken together, these results indicate that activation of the ASK1/p38 MAPK/p47phox cascade plays a central role in PPD/TLR2-induced ROS generation and suggests the existence of a 'ROS/ASK1' inflammatory amplification feedback loop in monocytes/macrophages. The altered regulation of this axis with an increasing free-radical burden may contribute to the immunopathogenesis of human TB.