Isolation of the rapidly activating delayed rectifier potassium current (I_Kr) from other cardiac currents has been a difficult task for quantitative study of this current. The present study was designed to separate I_Kr using Cs⁺ in cardiac myocytes. Cs⁺ have been known to block a variety of K⁺ channels, including many of those involved in the cardiac action potential such as inward rectifier potassium current I_K1 and the transient outward potassium current I_to. However, under isotonic Cs⁺ conditions (135 mM Cs⁺), a significant membrane current was recorded in isolated rabbit ventricular myocytes. This current displayed the voltage-dependent onset of and recovery from inactivation that are characteristic to I_Kr. Consistently, the current was selectively inhibited by the specific I_Kr blockers. The biophysical and pharmacological properties of the Cs⁺-carried human ether-a-go-go-related gene (hERG) current were very similar to those of the Cs⁺-carried I_Kr in ventricular myocytes. The primary sequence of the selectivity filter in hERG was in part responsible for the Cs⁺ permeability, which was lost when the sequence was changed from GFG to GYG, characteristic of other, Cs⁺-impermeable K⁺ channels. Thus the unique high Cs⁺ permeability in I_Kr channels provides an effective way to isolate I_Kr current. Although the biophysical and pharmacological properties of the Cs⁺-carried I_Kr are different from those of the K⁺-carried I_Kr, such an assay enables I_Kr current to be recorded at a level that is large enough and sufficiently robust to evaluate any I_Kr alterations in native tissues in response to physiological or pathological changes. It is particularly useful for exploring the role of reduction of I_Kr in arrhythmias associated with heart failure and long QT syndrome due to the reduced hERG channel membrane expression.