Introduction: Studies on isolated cardiomyocytes and ventricular wedges have shown contradicting data on the presence of transmural repolarization gradients in the left ventricle (LV). Here, we investigate electrophysiological heterogeneities of the canine LV free wall, using myocardial slices. Methods: Vibratome-cut myocardial slices (300 μm thick) were prepared from transmural LV biopsies (10x10x9 mm; n=4 hearts), tangentially to the wall surface. Multiphoton microscopy was used to visualise muscle fiber orientation. Multi-electrode arrays (60 microelectrodes, total area 4.9×4.9 mm) were used to map electrophysiological parameters. Preparations were paced at cycle lengths (CL) ranging from 1 to 4s. Values are means ± S.E.M. Results: Slices had longitudinal muscle fiber orientation, independent from transmural location. Activation-recovery intervals (ARI; Fig) correlated with transmural site at all pacing CL (p<0.001; ANOVA), with the longest values found in a 1.2 mm layer of the deep midmyocardium (4.8 to 6 mm from the epicardial surface; total LV thickness 9 mm). Longer pacing CL heterogeneously prolonged ARI (Fig) and increased transmural repolarization gradient from 63±7 to 106±3 ms (1s versus 4s CL; p<0.01 t-test). In plane ARI dispersion ranged from 9±1 to 18±3 ms, and was independent from transmural location and CL (p>0.05; ANOVA), suggesting the presence of electrophysiologically homogeneous and well coupled cells in each slice. Conclusion: The deep midmyocardium contains a well-defined homogeneous region of prolonged ARI, which may contribute to repolarisation gradients in the canine LV.