We have validated a new test for detecting Mycobacterium tuberculosis infection. A pool of synthetic peptides derived from ESAT-6 and CFP-10 proteins was used to detect the number of specific gamma interferon-producing T cells by means of an enzyme-linked immunospot assay. Sixty-eight individuals positive for M. tuberculosis infection, either human immunodeficiency virus-seropositive or -seronegative, were studied. The test results were highly specific (87.5%) and sensitive (93.1%), more so than a classical lymphoproliferative assay (specificity and sensitivity of 77.27%), opening new possibilities for diagnosis and screening of tuberculosis. Moreover, the test allowed us to distinguish individuals infected with M. tuberculosis from those vaccinated with BCG.