Regional variations in the function of ion channels and Ca²⁺ handling proteins underlie action potential waveform heterogeneity across the left ventricular wall. However, the functional impact of these electrophysiological differences on excitation-contraction (EC) coupling is incompletely understood. We used patch-clamp electrophysiology in combination with epifluorescence and confocal microscopy to examine EC coupling in mouse left ventricular epicardial (EPI) and endocardial (ENDO) cells. Consistent with previous work, we found that action potential duration at 90 % was significantly longer in ENDO (93.1 ± 5.4 ms (mean ± SEM), n=7) than in EPI (43.8 ± 6.1 ms, n=8, p<0.05) myocytes. In addition, our data indicated that ENDO cells had a higher systolic [Ca²⁺]_i (866 ± 88 nm, n=7) than EPI cells (506 ± 91 nm, n=8, p<0.05). L-type Ca²⁺ channel currents were similar in EPI and ENDO cells. Interestingly, SR Ca²⁺ load was higher in field stimulated ENDO (1337 ± 76 nm, n = 10) than in EPI cells (680 ± 23 nm, n-20, p <0.05) as was spontaneous Ca²⁺ spark activity (1.68+/-0.24 sparks(100 μm)^-1s^-1, Endo, n=114, versus 0.59 ± 0.15 sparks (100 μm)^-1s^-1, Epi, n=47, p<0.05). Furthermore, we found that the amplitude of the evoked whole-cell [Ca²⁺]_i transients was larger in ENDO than in EPI cells even if these cells were depolarized with identical waveforms. Taken together, our data are consistent with the view that transmural differences in EC coupling are produced by regionally distinct patterns of electrical activity and differences in the function of Ca²⁺ signaling proteins (e.g. Ryanodine receptors). Further investigation and clear characterization of the underlying mechanisms responsible for these differences in Ca2+ signaling are likely to provide greater understanding and insight into distinct regional differences and localization of signaling pathways in the heart.