A new ligand of the K+ channels sensitive to KTX was purified from the venom of Buthus occitanus tunetanus, using two steps of high-performance-liquid-chromatography and by following its ability to compete with [125I]-KTX for binding to the KTX receptor on rat brain synaptosomes. Amino-acid analysis, amino acid sequencing and mass spectroscopy defined this new ligand. KTX3, as a 37-amino acid peptide, with three disulfide bridges. Its sequence shares 76% identity with KTX. The main differences between the two peptides are in the N-terminal region and the residue position 34 located in the region involved in channel recognition. These differences may explain the 5-fold lower binding affinity of KTX3, IC50=50 pM, than KTX to rat brain synaptosomes. Specific antibodies raised against KTX (1-37) were not able to recognize KTX3.