The first example of a new sub-family of toxins (alpha-KTx20.1) from the scorpion Tityus trivittatus was purified, sequenced and characterized physiologically. It has 29 amino acid residues, three disulfide bridges assumed to adopt the cysteine-stabilized alpha/beta scaffold with a pI value of 8.98. The sequence identities with all the other known alpha-KTx are less than 40%. Its effects were verified using seven different cloned K(+) channels (vertebrate Kv1.1-1.5, Shaker IR and hERG) expressed in Xenopus leavis oocytes. The toxin-induced effects show large differences among the different K(+) channels and a preference towards Kv1.3 (EC50=7.9+/-1.4 nM).