2,3-Di-O-acyl-trehalose (DAT) is a glycolipid located on the outer layer of the Mycobacterium tuberculosis cell envelope. Due to its noncovalent linkage to the mycobacterial peptidoglycan, DAT could easily interact with host cells located in the focus of infection. The aim of the present work was to study the effects of DAT on the proliferation of murine spleen cells. DAT was purified from reference strains of M. tuberculosis, or M. fortuitum as a surrogate source of the compound, by various chromatography and solvent extraction procedures and then chemically identified. Incubation of mouse spleen cells with DAT inhibited in a dose-dependent manner concanavalin A-stimulated proliferation of the cells. Experiments, including the propidium iodide exclusion test, showed that these effects were not due to death of the cells. Tracking of cell division by labeling with 5,6-carboxyfluorescein diacetate succinimidyl ester revealed that DAT reduces the rounds of cell division. Immunofluorescence with an anti-CD3 monoclonal antibody indicated that T lymphocytes were the population affected in our model. Our experiments also suggest that the extent of the suppressive activity is strongly dependent on the structural composition of the acyl moieties in DATs. Finally, the inhibitory effect was also observed on antigen-induced proliferation of mouse spleen cells specific for Toxoplasma gondii. All of these data suggest that DAT could have a role in the T-cell hyporesponsiveness observed in chronic tuberculosis.