We investigated the action of TsTX‐K α on cloned Kv1.3 channels of the Shaker subfamily of voltage‐gated potassium channels, using the voltage–clamp technique. Highly purified TsTX‐K α was obtained from the venom of the Brazilian scorpion Tityus serrulatus using a new purification protocol. Our results show that TsTX‐K α blocks Kv1.3 with high affinity in two expression systems. TsTX‐K α blockade of Kv1.3 channels expressed in Xenopus oocytes was found to be completely reversible and to exhibit a pH dependence. The K D was 3.9 n M at pH 7.5, 9.5 n M at pH 7.0 and 94.5 n M at pH 6.5. The blocking properties of TsTX‐K α in a mammalian cell line (L929), stably transfected to express Kv1.3, were studied using the patch–clamp technique. In this preparation, the toxin had a K D of 19.8 n M at pH 7.4. TsTX‐K α was found to affect neither the voltage‐dependence of activation, nor the activation and deactivation time constants. The block appeared to be independent of the transmembrane voltage and the toxin did not interfere with the C‐type inactivation process. Taken as a whole, our findings indicate that TsTX‐K α acts as a simple blocker of Kv1.3 channels. It is concluded that this toxin is a useful tool for probing not only the physiological roles of Kv1.2, but also those mediated by Kv1.3 channels. British Journal of Pharmacology (2003) 139 , 1180–1186. doi: 10.1038/sj.bjp.0705343