To characterize the antigenic parts of the 16K protein of Mycobacterium tuberculosis, overlapping peptides according to the amino acid sequence of the 16K protein were synthesized. In total, 14 peptides of 20 amino acids in length with an overlap of 10 amino acids and two additional decapeptides (amino acids 31-40 and 61-70) were tested with eight anti-16K MoAbs and human sera. The common recognition site of MoAbs F67-8 and F67-16 was LRPTFDTRLM (amino acids 31-40) and of MoAbs F159-1 and F159-11 DPDKDVDIMV (amino acids 61-70). However, for binding of the MoAbs to these peptides additional amino acids were required at either the N- or C-terminus, suggesting that some kind of conformation is required. The recognition sites of the MoAbs F23-41, F23-49, F24-2 and TB68 could not be identified using the peptides, indicating that the MoAbs only bound to conformational epitopes and not to peptides which may contain parts of these epitopes. The MoAbs bound to beta-galactosidase fusion proteins comprising parts of the 16K protein, indicating that some kind of native conformation is present on the recombinant proteins. Sera from 14 of 19 patients with tuberculosis and none from 19 controls reacted with the purified 16K protein. Sera from four of these 14 patients reacted with two overlapping peptides (amino acids 71-100). Apparently, antibodies in patients' sera against the 16K protein are predominantly directed against conformational epitopes.