Novel diagnostic tools are needed to diagnose latent infection and to provide biologically meaningful surrogate markers to define cellular immune responses against Mycobacterium tuberculosis (MTB). Interferon gamma-based assays have recently been developed in addition to the more than 100-year-old tuberculin skin test (TST) for the immune diagnosis of MTB in blood. The advent of soluble MHC/peptide tetramer molecules allows to objectively enumerate antigen-specific T cells. We identified novel MHC class II-restricted MTB epitopes and used HLA-DR4 tetrameric complexes to visualize ex vivo CD4(+) T cells directed against the antigens Ag85B and the 19-kDa lipoprotein, shared between MTB and other Mycobacterium species, and CD4(+) T cells which recognize the MTB-associated ESAT-6 antigen. MTB-reactive CD4(+) T cells reside predominantly in the CD45RA(+) CD28(+) and CD45(-) CD28(+) T-cell subset and recognize naturally processed and presented MTB epitopes. HLA-DR4-restricted, Ag85B or ESAT-6-specific CD4(+) T cells show similar dynamics over time in peripheral blood mononuclear cells (PBMC) when compared with CD8(+) T cells directed against the corresponding HLA-A2-presented MTB epitopes in patients with pulmonary MTB infection and subsequent successful therapy. This was not found to be true for T-cell responses directed against the 19-kDa lipoprotein. The dissection of the cellular immune response in M. tuberculosis infection will enable novel strategies for monitoring MTB vaccine candidates and to gauge CD4(+) T cells directed against MTB.