omega-Agatoxin IVA, isolated from the venom of funnel web spider Agelenopsis aperta, blocks potently and selectively P-type calcium channels. This toxin, composed of 48 amino acids and containing 8 cysteine residues, was synthesized by the solid-phase procedure. The Cys residues were protected by acetamidomethyl (Acm) groups which were removed by mercuric acetate. During treatment with mercuric acetate, a by-product was detected, involving modification of tryptophan residues by the Acm groups. This side reaction can be completely prevented by addition of an excess of tryptophan in the reaction medium during Acm deprotection. The resulting peptide was submitted to an oxidative refolding, in different conditions, in order to determine the most favourable protocol. After formation of the four disulphide bonds, the toxin was purified by successive preparative HPLC, on two different supports, and fully characterized by analytical HPLC, capillary electrophoresis, amino acid analysis, mass spectrometry and Edman degradation. It was found to block the P-type calcium channel with a similar biological potency as described for the natural product.