We report here the total synthesis of the alpha-amylase inhibitor (AAI), a 32-residue-long peptide with three disulfide bridges, isolated from amaranth seeds (Chagolla-Lopez, A., Blanco-Labra, A., Patthy, A., Sanchez, R. & Pongor S. (1994) J. Biol. Chem. 269, 23675-23680). The synthesis was carried out using a stepwise solid-phase approach based on the Fmoc/t-Bu chemistry, combined with the S-acetamidomethyl protection for cysteines. The linear, reduced peptide was obtained after two reduction steps, using 1,4-dithio-DL-threitol and tri(2-carboxyethyl)phosphine hydrochloride in basic and acidic conditions, respectively. Disulfide bridges were formed by oxidative folding in a cystine/cysteine redox buffer, these conditions were found superior to air oxidation and to glutathione-catalyzed oxidative folding. The physiochemical and enzyme inhibitory properties of synthetic AAI were found identical with those of natural product. Several orthogonal protection schemes proved unsuccessful in obtaining a biologically active product.