We studied the action potential duration (APD) of isolated ventricular muscle fibers. Action potentials were recorded from four different sites (endocardium and epicardium at the apex and the base) in the canine left ventricle by microelectrode techniques. Action potentials from endocardial and corresponding epicardial specimens, and the differential potential between them were recorded simultaneously. The APDs at 60% and 90% repolarization (APD₆₀ and APD_9O) were measured during superfusion with normal (K⁺=2.7mM) and altered (K⁺=5.4mM) Tyrode's solutions. Results were: 1) the distributions of APDs agreed with previous descriptions from in vivo studies; 2) endo-epicardial APD gradients were different between the apex and the base, and apicobasal APD gradients were different between endocardium and epicardium; 3) the shapes of the differential potentials in normal and altered Tyrode's solution were similar to the electrocardiographic T wave form in normal and in mild hyperkalemia. It was concluded that nonhomogeneous APDs may be due to the inherent characteristics of cardiac muscle and the nonuniform endo-epicardial APD gradient helps to account for the genesis of the electrocardiographic T wave form.