Trehalose 6,6'-dimycolate (TDM) is a glycolipid component of the mycobacterial cell wall that causes immune responses in mice similar to Mycobacterium tuberculosis (MTB) infection, including granuloma formation with production of proinflammatory cytokines. The precise roles of tumour necrosis factor (TNF)-alpha, complement C5 and interleukin (IL)-6 in the molecular events that lead to the initiation and maintenance of the granulomatous response to TDM have not been fully elucidated. Macrophage proinflammatory responses from wild-type and complement-deficient mice after infection with MTB were assessed, and compared to responses from organisms in which surface TDM had been removed. Removal of TDM abolished proinflammatory responses, markedly so in the complement-deficient macrophages. Mice deficient in TNF-alpha, C5a and IL-6, along with wild-type C57BL/6 controls, were intravenously injected with TDM in a water-in-oil emulsion, and analysed for histological response and cytokine production in lungs. Wild-type C57BL/6 mice formed granulomas with increased production of IL-1beta, IL-6, TNF-alpha, macrophage inflammatory protein-1alpha (MIP-1alpha), IL-12p40, interferon-gamma (IFN-gamma), and IL-10 protein and mRNA. TNF-alpha-deficient mice failed to produce a histological response to TDM, with no increases in cytokine production following TDM administration. While C5a-deficient mice exhibited inflammation, they did not form structured granulomas and initially had decreased production of proinflammatory mediators. IL-6-deficient mice initiated granuloma formation, but failed to maintain the granulomas through day 7 and demonstrated decreased early production of proinflammatory mediators in comparison to wild-type mice. These data suggest that TNF-alpha is critical for initiation of the granulomatous response, C5a is necessary for formation of cohesive granulomas, and IL-6 plays a key role in the granuloma maintenance response to mycobacterial TDM.