The 39 amino acid peptide, margatoxin (MgTX), a potent inhibitor of the voltage-activated potassium channel (Kv 1.3) in human T lymphocytes, was synthesized by a solid phase technique. Formation of the disulfide bridges was rapid at pH 8.2. The final product was purified to homogeneity and was physically and biologically indistinguishable from the toxin prepared biosynthetically. The disulfide bridge pairing was similar to that found previously for the related toxin-charybdotoxin (3): from Cys7 to Cys29, from tested for inhibition of 125I margatoxin binding to voltage-activated potassium channels. The results indicate that the three C-terminal residues of MgTX are important for the efficient toxin binding to Kv1.3.