Recently a putative K+channel with homology to theShakerfamily of potassium channels has been cloned from human ventricular myocardium. However, proof that the cDNA encodes a K+channel requires appropriate translation and expression of a functional ion-selective channel. Therefore, expression of this putative human K+channel DNA was attempted by cytoplasmic injections of in vitro transcribed cRNA intoXenopus laevisoocytes and screening by two-electrode voltage-clamp methods. This resulted in expression of voltage-gated channels that rapidly inactivated (time constant of inactivation, 47.6±3.6 msec; 0 mV; n=10) and were at least 50 times more selective for K+than Na+(Na+/K+permeability ratio of 0.02). The channels showed voltage-dependent activation (half-maximal voltage, −34±0.7 mV; n=5), and 50% of the channels were inactivated within 2 seconds when the membrane potential was clamped near −60 mV (half-maximal voltage, −62±7 mV; n=10). The expressed protein resulted in a K+current that had many properties similar to the 4-aminopyridine-sensitive calcium-insensitive component of the cardiac transient outward current that is observed in native cardiac myocytes and thus may serve as one molecular substrate for this current.