Although electrical heterogeneity within the ventricular myocardium has been the focus of numerous studies, little attention has been directed to the mechanical correlates. This study examines unloaded cell shortening, Ca(2+) transients, and inward L-type Ca(2+) current (I(Ca,L)) characteristics of epicardial, endocardial, and midmyocardial cells isolated from the canine left ventricle. Unloaded cell shortening was recorded using a video edge detector, Ca(2+) transients were measured in cells loaded with 15 microM fluo-3 AM and voltage and current-clamp recordings were obtained using patch-clamp techniques. Time to peak and latency to onset of contraction were shortest in epicardial and longest in endocardial cells; midmyocardial cells displayed an intermediate time to peak. When contraction was elicited using uniform voltage-clamp square waves, epicardial versus endocardial distinctions persisted and midmyocardial cells displayed a time to peak comparable to that of epicardium. The current-voltage relationship for I(Ca,L) and fluorescence-voltage relationship were similar in the three cell types when quantitated using square pulses. However, peak I(Ca,L) and total charge were significantly larger when an epicardial versus endocardial action potential waveform was used to elicit the current under voltage-clamp conditions. Sarcoplasmic reticulum Ca(2+) content, assessed by rapid application of caffeine, was largest in epicardial cells and contributed to a faster time to peak. Our data point to important differences in calcium homeostasis and mechanical function among the three ventricular cell types. These differences serve to synchronize contraction across the ventricular wall. Although these distinctions are conferred in part by differences in electrical characteristics of the three cell types, intrinsic differences in excitation-contraction coupling are evident.