The venom of the scorpion, Tityus serrulatus, was fractionated to investigate the chemical and pharmacological properties of its alpha-toxin content. Three alpha-toxins (Ts III, Ts IV and Ts V) were purified by conventional chromatography (gel filtration and ion-exchange chromatography), followed by immunoaffinity chromatography. Competition experiments using reference alpha- and beta-toxins suggested that these alpha-toxins were contaminated with around 0.01% of beta-toxin. The sequence of the first 30 amino acids of Ts IV was established. Using an oligonucleotide probe, a cDNA encoding its precursor was cloned from a venom gland cDNA library. The primary structure deduced from the cDNA nucleotide sequence provides possible explanations for the polymorphism of these three molecules.