Spatial heterogeneity of the action potential and its influence on arrhythmia vulnerability is known. However, heterogeneity of intracellular calcium handling and, in particular, its effect on the electrophysiological substrate is less clear. Using optical mapping techniques, calcium transients and action potentials were recorded simultaneously from ventricular sites across the transmural wall of the arterially perfused canine left ventricular wedge preparation during steady-state baseline pacing and rapid pacing. During baseline pacing, the decay of intracellular calcium to diastolic levels and calcium transient duration were slower (70%, P<0.005) and longer (20%, P<0.005), respectively, closer to the endocardial surface compared with the epicardial surface. Tissue samples isolated from the left ventricular wall demonstrate that sarcoplasmic reticulum Ca2+ ATPase (SERCA2a) expression was significantly less in the subendocardial and midmyocardial layers compared with the subepicardial layer. In contrast, no significant difference in the transmural expression of Na+-Ca2+ exchanger was observed. During rapid pacing, calcium transient alternans and increased levels of diastolic intracellular calcium were significantly greater (P<0.01) closer to the endocardium (101%+/-62% and 41%+/-15%, respectively) compared with the epicardium (12%+/-7% and 12%+/-14%, respectively). In conclusion, cells closer to the endocardium exhibit a slower decay of intracellular calcium compared with cells near the epicardium, which may be due in part to reduced expression of SERCA2a. As a possible consequence, calcium transient alternans and increased diastolic levels of intracellular calcium may occur preferentially closer to the endocardial surface.