The race-specific peptide elicitor AVR9 of the fungal pathogen Cladosporium fulvum specifically induces a hypersensitive response in tomato genotypes carrying the complementary resistance gene Cf-9. The total chemical syntheses of this 28-residue AVR9 peptide containing three disulfide bonds, and of three mutant peptides [R8K]AVR9, [F10A]AVR9 and [F21A]AVR9, have been accomplished. The syntheses were carried out using a stepwise solid-phase approach based on tBoc chemistry. The disulfide bridges were formed by air oxidation. The correctness of the chemical structure of all folded synthetic peptides was confirmed by combined NMR and MS analyses. The biological activity and a number of physicochemical properties of folded synthetic AVR9 are identical to those of native fungal 28-residue AVR9. The overall conformations of the folded synthetic mutant peptides were comparable to that of synthetic wild-type AVR9 as demonstrated by NMR spectroscopy. Mutant [R8K]AVR9 showed a threefold higher, and mutant [F10A]AVR9 a threefold lower necrosis-inducing activity when compared to synthetic wild-type AVR9. However, mutant [F21A]AVR9 showed hardly any necrosis-inducing activity. Affinity for polyclonal antibodies raised against native fungal AVR9 is positively correlated with the necrosis-inducing activity of the synthetic AVR9 peptides ([R8K]AVR9 > wild-type AVR9 > [F10A]AVR9 > [F21A]AVR9).