Vaccination against and diagnosis of tuberculosis are still insufficient. Proteins secreted by Mycobacterium tuberculosis induce strong immune responses in tuberculosis and constitute prime candidates for development of novel vaccines against tuberculosis as well as for immunodiagnostic assays. We investigated the role of the secreted proteins MPT63, MPT64 and ESAT6 from M. tuberculosis in healthy individuals and tuberculosis patients. None of the secreted proteins stimulated peripheral blood mononuclear cells from healthy donors. In contrast, CD4+ T cells from many tuberculosis patients were stimulated in an MHC class II-restricted fashion by ESAT6, but not by MPT63 or MPT64. T cell reactivities of tuberculosis patients were focused on the N-terminal region of ESAT6. The ESAT6 T cell epitopes were presented by different HLA-DR phenotypes. Cell cultures responding to either ESAT6 or synthetic peptides thereof showed mRNA transcripts for macrophage inflammatory protein (MIP)-1alpha, monocyte chemotactic protein (MCP)-1 or IL-8 and production of IFN-gamma and MIP-1alpha. Our results suggest that the secreted M. tuberculosis proteins MPT63, MPT64 or ESAT6 do not stimulate unprimed T cells, and that ESAT6 may be a potential candidate antigen for detection of clinical disease.