This study examines the amplitude of sodium-calcium exchange current (I _NaCa) in epicardial, midmyocardial, and endocardial canine ventricular myocytes. Whole cell currents were recorded at 37^°C using standard or perforated-patch voltage-clamp techniques in the absence of potassium, calcium-activated chloride, and sodium-pump currents.I _NaCa was triggered by release of calcium from the sarcoplasmic reticulum or by rapid removal of external sodium.I _NaCa was large in midmyocardial myocytes and significantly smaller in endocardial myocytes, regardless of the method used to activate I _NaCa. I _NaCaat −80 mV was −0.316 ± 0.013, −0.293 ± 0.016, and −0.210 ± 0.007 pC/pF, respectively, in midmyocardial, epicardial, and endocardial myocytes when activated by the calcium transient. When triggered by sodium removal, peakI _NaCa was 0.74 ± 0.04, 0.57 ± 0.04, and 0.50 ± 0.03 pA/pF, respectively, in midmyocardial, epicardial, and endocardial myocytes. Epicardial I _NaCa was smaller than midmyocardial I _NaCa when activated by removal of external sodium but was comparable to epicardial and midmyocardial I _NaCa when activated by the normal calcium transient, implying possible transmural differences in excitation-contraction coupling. Our results suggest thatI _NaCa differences contribute to transmural electrical heterogeneity under normal and pathological states. A large midmyocardial I _NaCa may contribute to the prolonged action potential of these cells as well as to the development of triggered activity under calcium-loading conditions.