Human T cell responses to ESAT-6 and eight synthetic overlapping peptides were investigated in tuberculosis (TB) patients and control subjects from regions of high and low endemicity for TB. ESAT-6 was recognized by 65% of all tuberculin purified protein derivative-responsive TB patients, whereas only 2 of 29 bacille Calmette-Guérin-vaccinated Danish healthy donors recognized this molecule. In Ethiopia, a high frequency (58%) of healthy contacts of TB patients recognized ESAT-6. All of the peptides were recognized by some donors, indicating that the molecule holds multiple epitopes. Danish and Ethiopian patients differed in the fine specificity of their peptide responses. Recognition of the C-terminal region (aa 72-95) was predominant in Danish patients, whereas recognition of aa 42-75 was predominant in Ethiopia. The relationship of these differences to the distribution of HLA types in the two populations is discussed. This study demonstrates that ESAT-6 is frequently recognized during early infection and holds potential as a component of a future TB-specific diagnostic reagent.