AbstractERG potassium channels specify one component of the delayed rectifier in the heart and are likely to play an important functional role in other excitable cells. Compared to other K+ channels, the human ERG (hERG) channel possesses an unusually long S5‐P linker that presumably forms an alpha‐helix important for channel function. hERG‐specific toxins bind to the outer mouth of the hERG channel. Channel residues in the middle of the S5‐P linker and at the pore entrance are critical for toxin binding. One of these scorpion toxins is BeKm‐1. Residues critical for BeKm‐1 binding to the hERG channel are located in the alpha‐helix and the following loop, whereas the ‘traditional’ interaction surface of other short scorpion toxins is formed by residues on the β‐sheet. This unique localization of BeKm‐1's interaction surface and its specific action on the hERG channel suggest a unique outer mouth structure of the hERG channel. We used the mutant cycle analysis approach to define contacts in the toxin–channel complex. This information provides critical constraints and is important for molecular modeling of the hERG pore structure. Copyright © 2004 John Wiley & Sons, Ltd.