© 2016 The Authors. In the heart, the left ventricle pumps blood at higher pressure than the right ventricle. Within the left ventricle, the electromechanical properties of ventricular cardiac myocytes vary transmurally and this may be related to the gradients of stress and strain experienced in vivo across the ventricular wall. Diabetes is also associated with alterations in hemodynamic function. The aim of this study was to investigate shortening and Ca2+ transport in epicardial (EPI) and endocardial (ENDO) left ventricular myocytes in the streptozotocin (STZ)-induced diabetic rat. Shortening, intracellular Ca2+ and L-type Ca2+ current (ICa,L) were measured by video detection, fura-2 microfluorimetry, and whole-cell patch clamp techniques, respectively. Time to peak (TPK) shortening was prolonged to similar extents in ENDO and EPI myocytes from STZ-treated rats compared to ENDO and EPI myocytes from controls. Time to half (THALF) relaxation of shortening was prolonged in ENDO myocytes from STZ-treated rats compared to ENDO controls. TPK Ca2+ transient was prolonged in ENDO myocytes from STZ-treated rats compared to ENDO controls. THALF decay of the Ca2+ transient was prolonged in ENDO myocytes from STZ-treated rats compared to ENDO controls. Sarcoplasmic reticulum (SR) fractional release of Ca2+ was reduced in EPI myocytes from STZtreated rats compared to EPI controls. ICa,L activation, inactivation, and recovery from inactivation were not significantly altered in EPI and ENDO myocytes from STZ-treated rats or controls. Regional differences in Ca2+ transport may partly underlie differences in ventricular myocyte shortening across the wall of the healthy and the STZ-treated rat left ventricle.