Chlorotoxin (CTX) is a 36–amino acid peptide with 8 Cys residues that forms four Cys-Cys bonds. It has high affinity for the glioma-specific chloride channel and matrix metalloprotease-2. Structural and binding properties of CTX analogs with various Cys residue substitutions with L-a-aminobutyric acid (Abu) have been previously reported. Using 4.2 ìs molecular dynamics, we compared the conformational and essential space sampling of CTX and analogs [Abu/Ser2,19]CTX, [Abu/Ser5,28]CTX, [Abu/Ser16,33]CTX, [Abu/Ser20,35]CTX, and [Abu/Ser2,5,16,19,20,28,33,35]CTX. The native and substituted peptides maintained a high degree of a-helix propensity from residues 8 through 21, with the exception of [Ser5,28]CTX and [Abu16,33]CTX. In agreement with previous circular dichroism spectropolarimetry results, the C-terminal b-sheet content varied less from residues 25 through 29 and 32 through 36 and was well conserved in all analogs, except: [Abu16,33]CTX, [Ser20,35]CTX, [Abu2,5,16,19,20,28,33,35]CTX, and [Ser2,5,16,19,20,28,33,35]CTX. The Cys16-Cys33 and Cys20-Cys35 Cys-Cys bonds appear to be required to maintain the ab-motif of CTX. Their selective substitution with Ser16,33 however, may mitigate the destabilizing effect of Cys16-Cys33 substitution by the formation of an inter residue H-bond from OgH of Ser16 to OgH of Ser33 bridged by a water molecule. All peptides shared considerable sampled conformational space, which explains the retained receptor binding of the nonnative analogs.