Mice treated with viable Mycobacterium tuberculosis with no glycolipid trehalose dimycolate (TDM) on the outer cell wall (delipidated M. tuberculosis) by intraperitoneal or intratracheal inoculation presented an intense recruitment of polymorphonuclear cells into the peritoneal cavity and an acute inflammatory reaction in the lungs, respectively. In addition, lung lesions were resolved around the 32nd day after intratracheal inoculation. TDM-loaded biodegradable poly-DL-lactide-coglycolide microspheres as well as TDM-coated charcoal particles induced an intense inflammatory reaction. In addition, high levels of interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-alpha), IL-12, IL-10, gamma interferon (IFN-gamma), and IL-4 production were detected in lung cells, and nitric oxide (NO) production was high in culture supernatants of bronchoalveolar lavage cells. These in vivo data were confirmed by in vitro experiments using peritoneal macrophages cultured in the presence of TDM adsorbed onto coverslips. High levels of IFN-gamma, IL-6, TNF-alpha, IL-12, IL-10, and NO were detected in the culture supernatants. Our results suggest that TDM contributes to persistence of infection through production of cytokines, which are important for the recruitment of inflammatory cells and maintenance of a granulomatous reaction. In addition, our findings are important for a better understanding of the immunostimulatory activity of TDM and its possible use as an adjuvant in experiments using DNA vaccine or gene therapy against tuberculosis.