T cell responses to ESAT-6 and culture filtrate protein 10 (CFP-10), antigens expressed by Mycobacterium tuberculosis but not by M. bovis bacille Calmette-Guérin (BCG), were found to discriminate reliably between infection with M. tuberculosis and BCG vaccination. Because the esat-6 and cfp-10 genes occur in M. kansasii and M. marinum, T cell responses to ESAT-6 and CFP-10 were investigated in patients infected with M. kansasii or M. marinum, persons intensively exposed to environmental mycobacteria, and unexposed control subjects. Tuberculin skin tests were performed, and peripheral blood mononuclear cells were cocultured with ESAT-6, CFP-10, peptide mixtures of ESAT-6 and CFP-10, and control antigens. When enzyme-linked immunosorbent assay (ELISA) and enzyme-linked immunospot assay (ELISPOT) were used to measure interferon-gamma production, most M. kansasii- or M. marinum-infected patients and several persons exposed to environmental mycobacteria were found to respond to ESAT-6 and/or CFP-10. ELISA and ELISPOT yielded comparable results, as did whole antigen and peptides (P<.0001). These results may be relevant for the development of novel assays for diagnosis of tuberculosis.