Charybdotoxin is a 37-residue polypeptide toxin from scorpion venom, which acts by blocking voltage-gated and Ca 2 + -activated K + channels. We have synthesized charybdotoxin and three mono-substituted analogues using an Fmoc-tBu protocol. The Phe-2 → Tyr analogue was chosen to introduce a site for Tyr iodination which was distinct from the K + channel binding surface, while the Glu-12 → Gln and Arg-19 → His analogues were studied to probe the roles of charged residues at these positions in the structure and activity of the toxin. The synthetic native molecule was equipotent with natural toxin in inhibiting the human erythrocyte Ca 2 + -dependent K + channel. The affinities of all three analogues for the erythrocyte K + channel were slightly reduced, with the Arg-19 → His analogue showing the greatest increase in IC 5 0 (2.3-fold). Two-dimensional 1 H-NMR studies of these analogues showed that all three had structures very similar to those of the native molecule. The most significant perturbation was associated with the Glu-12 to Gln substitution, which appeared to destabilise the N-terminal half of the α-helix, possibly due to the weakening of an N-terminal helix capping interaction which is apparent from our NMR data. His-21 has a pK a more than one unit below the value for a non-interacting histidine. Possible reasons for this are that the imidazolium side chain is partly buried and is located near positively charged moieties. Thus, His-21 would be neutral at physiological pH, where charybdotoxin binds to the potassium channel.