The 16-kDa protein of Mycobacterium tuberculosis provokes specific immune responses; it is thus a target for the development of peptide-based diagnostic reagents and subunit vaccines. Previous studies have demonstrated the presence of several regions containing murine and human T-cell epitopes. Within the 91-110 immunodominant domain, we found that peptides comprising the sequence of 91SEFAYGSFVRTVSL104 elicit specific T-cell responses in both human T-cell clones and human peripheral blood mononuclear cells (PBMC) from PPD+ (purified protein derivative) individuals. Elongation of this peptide towards the C-terminal end did not provide more effective peptides, but the removal of residue 91Ser resulted in an almost complete loss of functionality. However, the introduction of an acetyl group at the N-terminal of residue 92Glu produced a shorter peptide (Ac-92EFAYGSFVRTVSL104) exhibiting properties required for efficient T-cell responses. CD measurements indicated that peptide 91SEFAYGSFVRTVSLPVGADE110 adopts a helical conformation in trifluoroethanol. We found that the N-terminal part of this sequence plays a major role in the induction of proliferative T-cell responses and is responsible for the highly ordered, helical secondary structure. The "lead" structure described here could also be considered in the development of synthetic peptides or multicomponent peptide mixtures for the early detection, monitoring, or preventing Mycobacterium tuberculosis infection with optimized T-cell response-provoking capacity.