We previously purified and characterized a peptide toxin, birtoxin, from the South African scorpion Parabuthus transvaalicus. Birtoxin is a 58-residue, long chain neurotoxin that has a unique three disulfide-bridged structure. Here we report the isolation and characterization of ikitoxin, a peptide toxin with a single residue difference, and a markedly reduced biological activity, from birtoxin. Bioassays on mice showed that high doses of ikitoxin induce unprovoked jumps, whereas birtoxin induces jumps at a 1000-fold lower concentration. Both toxins are active against mice when administered intracerebroventricularly. Mass determination indicated an apparent mass of 6615 Da for ikitoxin vs. 6543 Da for birtoxin. Amino acid sequence determination revealed that the amino-acid sequence of ikitoxin differs from birtoxin by a single residue change from glycine to glutamic acid at position 23, consistent with the apparent mass difference of 72 Da. This single-residue difference renders ikitoxin much less effective in producing the same behavioral effect as low concentrations of birtoxin. Electrophysiological measurements showed that birtoxin and ikitoxin can be classified as beta group toxins for voltage-gated Na+ channels of central neurons. It is our conclusion that the N-terminal loop preceding the alpha-helix in scorpion toxins is one of the determinative domains in the interaction of toxins with the target ion channel.