The Kv2.1 voltage-activated K+ channel, a Shab-related K+ channel isolated from rat brain, is insensitive to previously identified peptide inhibitors. We have isolated two peptides from the venom of a Chilean tarantula, G. spatulata, that inhibit the Kv2.1 K+ channel. The two peptides, hanatoxin, (HaTx1) and hanatoxin2 (HaTx2), are unrelated in primary sequence to other K+ channel inhibitors. The activity of HaTx was verified by synthesizing it in a bacterial expression system. The concentration dependence for both the degree of inhibition at equilibrium (Kd = 42 nM) and the kinetics of inhibition (kon = 3.7 × 104 M−1s−1; koff = 1.3 × 10−3 s−1), are consistent with a bimolecular reaction between HaTx and the Kv2.1 K+ channel. Shaker-related, Shaw-related, and eag K+ channels were relatively insensitive to HaTx, whereas a Shal-related K+ channel was sensitive. Regions outside the scorpion toxin binding site (S5-S6 linker) determine sensitivity to HaTx. HaTx introduces a new class of K+ channel inhibitors that will be useful probes for studying K+ channel structure and function.