Cardiac function is known to be impaired in diabetes. Alterations in intracellular calcium handling have been suggested to play a pivotal role. This study aimed to test the hypothesis that ß-adrenergic activation can reveal the functional derangements of intracellular calcium handling of the 4-week diabetic heart. Langendorff perfused hearts of 4-week streptozotocin-induced diabetic rats were subjected to the ß-adrenoceptor agonist isoproterenol. Cyclic changes in [Ca2+]i levels were measured throughout the cardiac cycle using Indo-1 fluorescent dye. Based on the computational analysis of the [Ca2+]i transient the kinetic parameters of the sarcoplasmic reticulum Ca2+-ATPase and the ryanodine receptor were determined by minimizing the squared error between the simulated and the experimentally obtained [Ca2+]i transient. Under unchallenged conditions, hemodynamic parameters were comparable between control and diabetic hearts. Isoproterenol administration stimulated hemodynamic function to a greater extent in control than in diabetic hearts, which was exemplified by more pronounced increases in rate of pressure development and decline. Under unchallenged conditions, [Ca2+]i amplitude and rate of rise and decline of [Ca2+]i as measured throughout the cardiac cycle were comparable between diabetic and control hearts. Differences became apparent under ß-adrenoceptor stimulation. Upon ß-activation the rate-pressure product showed a blunted response, which was accompanied by a diminished rise in [Ca2+]i amplitude in diabetic hearts. Computational analysis revealed a reduced function of the sarcoplasmic reticulum Ca2+-ATPase and Ca2+-release channel in response to ß-adrenoceptor challenge. Alterations in Ca2+i handling may play a causative role in depressed hemodynamic performance of the challenged heart at an early stage of diabetes.