1. Pyruvate has been shown to enhance the contractile performance of cardiac muscle when provided as an alternative substrate to glucose. The aims of the present study were to determine whether the inotropic effects of pyruvate are due to increased mobilization of intracellular Ca2+ ([Ca2+]i) and to compare the effects of pyruvate on [Ca2+]i levels in myocytes from normal and diabetic animals. 2. Fura-2 was used to monitor [Ca2+]i in ventricular myocytes isolated from control and streptozotocin-treated male Wistar rats. The experiments were performed at 25 degrees C, with an extracellular [Ca2+] of 1.5 mmol/L and either 10 mmol/L glucose or 10 mmol/L pyruvate as the substrate. 3. In myocytes from both control and diabetic rats, increasing the stimulus frequency from 0.33 to 2.0 Hz resulted in significant increases in resting and peak [Ca2+]i as well as in the amplitude of the [Ca2+]i transient, irrespective of substrate. Compared with glucose, pyruvate significantly increased resting and peak [Ca2+]i and the amplitude of the [Ca2+]i transient at each stimulus frequency in myocytes from both control and diabetic animals. However, the extent of potentiation of the [Ca2+]i transient amplitude produced by pyruvate was significantly less in myocytes from the diabetic rats. 4. The rate of restitution of the [Ca2+]i transient was used as an index of the rate of Ca2+ cycling by the sarcoplasmic reticulum (SR). Pyruvate enhanced the rate of restitution in control but not diabetic rat cells. 5. The time course of decay of the [Ca2+]i transient was analysed as a measure of the rate of removal of Ca2+ from the cytosol. Pyruvate tended to increase the rate of decay in cells from control but not diabetic animals. The rate of decay was slower in cells from diabetic animals compared with controls. 6. The data reveal that pyruvate increases SR Ca2+ cycling, leading to greater Ca2+ release and an increase in the amplitude of the [Ca2+]i transient. Therefore, it seems highly likely that increased [Ca2+]i mobilization is responsible for the previously reported positive inotropic actions of pyruvate. These effects of pyruvate are attenuated in diabetic rat cells, which may reflect an impaired capacity of mitochondria in diabetic hearts to oxidize pyruvate, thus limiting potential energetic benefits.