The granulomatous response is the characteristic histological feature of Mycobacterium tuberculosis infection that is essential for organism containment. Trehalose 6,6-dimycolate (TDM), a cell-wall glycolipid present on most mycobacterial species, has been implicated in the pathogenesis of M. tuberculosis infection. TDM has potent immunoregulatory and inflammatory properties, and can be used to model granulomatous reactions that mimic, in part, pathology caused during active infection. This study examined the hypersensitive granulomatous response, focusing on cellular responses specific to TDM. Lungs from mice immunized with TDM emulsion demonstrated exacerbated histological damage, inflammation, and lymphocytic infiltration upon subsequent challenge with TDM. Splenocytes recovered from these mice demonstrated significant interferon (IFN)-gamma production during recall response to TDM, as well as increased production of proinflammatory mediators (tumour necrosis factor-alpha, interleukin-6 and macrophage inflammatory protein-1alpha). The exacerbated response could be adoptively transferred to naïve mice. Administration of non-adherent lymphocytes or purified CD3(+) cells from TDM-immunized mice led to increased inflammation, lymphocytic infiltration, and vascular endothelial cell damage upon challenge with TDM. Recipient mice that received immunized CD3(+) lymphocytes demonstrated significant increases in Th1-type cytokines and proinflammatory mediators in lung tissue following TDM challenge. When CD1d(-/-) mice were immunized with TDM, they failed to generate a specific IFN-gamma response, suggesting a role for this molecule in the generation of hypersensitivity. These experiments provide further evidence for the involvement of TDM-specific CD3(+) T cells in pathological damage elicited during M. tuberculosis infection.