Recent studies have described regional differences in the electrophysiology and pharmacology of ventricular myocardium in canine, feline, rat, guinea pig, and human hearts. In this study, we use standard microelectrode and whole-cell patch-clamp techniques to examine the characteristics of the action potential and the delayed rectifier K⁺ current (I_K) in epicardial, M region (deep subepicardial to midmyocardial), and endocardial cells isolated from the canine left ventricle. Cells from the M region displayed much longer action potential durations (APDs) at slow rates. At a basic cycle length of 4 s, APD measured at 90% repolarization was 358±16 (mean±SEM), 262±12, and 287±11 ms in cells from the M region, epicardium, and endocardium, respectively. Steady state APD-rate relations were steeper in cells from the M region. In complete Tyrode’s solution, I_K was smaller in myocytes from the M region when compared with those isolated from the epicardium or endocardium. Further characterization of I_K was conducted in a Na⁺-, K⁺-, and Ca²⁺-free bath solution to isolate the slowly activating component of the delayed rectifier (I_Ks) from the rapidly activating component (I_Kr). I_Ks was significantly smaller in M cells than in epicardial and endocardial cells. With repolarization to −20 mV, I_Ks tail current density was 1.99±0.30 pA/pF (mean±SEM) in epicardial cells, 1.83±0.18 pA/pF in endocardial cells, and 0.92±0.14 pA/pF in M cells. Voltage dependence and time course of activation and deactivation of I_Ks were similar in the three cell types. The relative contribution of I_Kr and I_Ks among the three cell types was examined by using 6 mmol/L [K⁺]_o Tyrode’s solution with and without E-4031, a highly selective blocker of I_Kr. An E-4031–sensitive current was observed in the presence but not in the absence of extracellular K⁺. This rapidly activating component showed characteristics similar to those of I_Kr as described in rabbit and cat ventricular cells. Deactivation of I_Kr was significantly slower than that of I_Ks. I_Kr (E-4031–sensitive component) tail current density was similar in the three cell types, whereas I_Ks (E-4031–insensitive component) tail current density was significantly smaller in the M cells. Our results suggest that the distinctive phase-3 repolarization features of M cells are due in part to a lesser contribution of I_Ks and that this distinction may also explain why M cells are the main targets for agents that prolong APD in ventricular myocardium. These findings may advance our understanding of the ionic basis for the electrocardiographic T wave, U wave, and long QT intervals as well as our understanding of factors contributing to the development of cardiac arrhythmias.