Chlorotoxin (CTX) is a 36- amino acid peptide isolated from the giant yellow Israeli scorpion Leiurus quinquestriarus¹. The structure of CTX comprises four disulfide bonds; three of the disulfide bonds are involved in linking an α-helix to a three-stranded antiparallel beta-sheet with the remaining disulfide bond connecting the N-terminal region with the rest of peptide². We have studied the role of the disulfide bonds by replacing each disulfide bond by a pair of L-α-aminobutyric acid (Abu). Five analogues were synthesised, using solid-phase synthesis, and named CTX1, CTX2, CTX3, CTX4 and CTX5. All Cys residues were replaced in CTX1, whereas one pair of Cys residues was replaced in each of the remaining four analogues. CTX2, CTX3, CTX4 and CTX5 have Abu residues in positions 2 and 19, 5 and 28, 16 and 33, and 20 and 35, respectively. Preliminary folding experiments of CTX2 to CTX5 in several oxidation buffers revealed that CTX2 and 3 showed a defined early-eluting peak on RP-HPLC, which could represent a folded molecule with the Cys residues arranged in the native disulfide connectivity. On the other hand, CTX4 and 5 did not yield a defined peak in the oxidation buffers, suggesting that the disulfide bonds which were mutated in those analogues are important for forming the native structure. Circular dichroism analysis showed that the conformation of CTX2 and 3 is similar to that of CTX but very different to that of CTX1. Thus, the cystine pairs 2 and 19 (CTX2) and 5 and 28 (CTX3) could be modified without significantly affecting the folding properties of the peptides. Furthermore, CTX1, CTX2 and CTX3 inhibited the migration of human umbilical vein endothelial cells (HUVECs) with the same potency as that of native CTX.These results could have interesting implications in protein design and drug development using chlorotoxin as a scaffold.