In cardiac myocytes the repolarizing potassium current results from the two currents I_Kr (Kv11.1) and I_Ks (Kv7.1/KCNE1). The concerted activity of these currents is crucial for the precise timing and efficiency of cardiac repolarization. Drug-induced current inhibition can cause ECG changes and sometimes potentially fatal arrhythmias. A novel compound reported to potently inhibit cardiac I_Ks channels is JNJ-303. This substance evoked unprovoked torsade de pointes (TdP) arrhythmias in vivo in an anaesthetized dog model. The aim of the present study was to gain deeper insights into the molecular mechanisms underlying JNJ-303 inhibition of I_Ks. Therefore, we expressed slowly activating Kv7.1/KCNE1 channels in Xenopus oocytes and tested their sensitivity to JNJ-303 using two electrode voltage clamp. To determine the putative binding site of this compound we used Alanine-scanning mutagenesis combined with voltage clamp analysis. Here, we report that JNJ-303 potently blocks Kv7.1/KCNE1 channels and modulates channel gating. We localized the putative drug receptor to the pore region and the S6 transmembrane segment of the channel complex. In the future the growing knowledge of inhibitor binding sites may allow us to design novel ion channel modulators and to prevent unwanted binding to channels like I_Kr or I_Ks during the development of drugs aimed for different indications.