Cardiac excitation-contraction (E-C) coupling abnormalities in chemically induced diabetes have been well defined. Heart dysfunction has also been reported in diabetes of genetic origin. The purpose of this study was to determine whether heart dysfunction in genetically predisposed diabetes is attributable to impaired E-C coupling at the cellular level. Myocytes were isolated from 1-yr-old BioBreed (BB) spontaneously diabetic-prone (BB/DP) rats and their diabetic-resistant littermates (BB/DR). Mechanical properties were evaluated by use of a video edge-detection system. Myocytes were electrically stimulated at 0.5 Hz. The contractile properties analyzed included peak shortening (PS), time-to-peak shortening (TPS), time-to-90% relengthening (TR₉₀), and maximal velocities of shortening and relengthening (±dL/dt). Intracellular Ca²⁺ handling was evaluated with fura 2 fluorescent dye. Myocytes from spontaneously diabetic hearts exhibited a depressed PS, prolonged TPS and TR₉₀, and reduced ±dL/dt. Consistent with the mechanical response, myocytes from the BB/DP group displayed reduced resting and peak intracellular Ca²⁺ concentration associated with a slowed Ca²⁺-transient decay. Furthermore, myocytes from BB/DP hearts were less responsive to increases in extracellular Ca²⁺ and norepinephrine and equally responsive to increases in stimulation frequency and KCl compared with those from the BB/DR group. These results suggest that the genetic diabetic state produces altered cardiac E-C coupling, in part, because of abnormalities of the myocyte, similar to that demonstrable after chemically induced diabetes or during human diabetes.