CD1-restricted lipid-specific T lymphocytes are primed during infection with Mycobacterium tuberculosis, the causative agent of tuberculosis. Here we describe the antigenicity of glycerol monomycolate (GroMM), which stimulates CD1b-restricted CD4(+) T cell clones. Chemical characterization of this antigen showed that it exists as two stereoisomers, one synthetic isomer being more stimulatory than the other. The hydroxyl groups of glycerol and the mycolic acid length are critical for triggering the T cell responses. GroMM was presented by M. tuberculosis-infected dendritic cells, demonstrating that the antigen is available for presentation during natural infection. Ex vivo experiments showed that GroMM stimulated T cells from vaccinated or latently infected healthy donors but not cells from patients with active tuberculosis, suggesting that GroMM-specific T cells are primed during infection and their detection correlates with lack of clinical active disease.