We have investigated the effects of acute acidosis on ventricular myocyte shortening and intracellular Ca²⁺ in streptozotocin (STZ)-induced diabetic rat. Shortening and intracellular Ca²⁺ were measured in electrically stimulated myocytes superfused with either normal Tyrode solution pH adjusted to either 7.4 (control solution) or 6.4 (acid solution). Experiments were performed at 35–36°C. At 8–12 weeks after treatment, the rats that received STZ had lower body and heart weights compared to controls, and blood glucose was characteristically increased. Contractile defects in myocytes from diabetic rat were characterized by prolonged time to peak shortening. Superfusion of myocytes from control and diabetic rats with acid solution caused a significant reduction in the amplitude of shortening; however, the magnitude of the response was not altered by STZ treatment. Acid solution also caused significant and quantitatively similar reductions in the amplitude of Ca²⁺ transients in myocytes from control and diabetic rats. Effects of acute acidosis on amplitude of myocyte contraction and Ca²⁺ transient were not significantly altered by STZ treatment. Altered myofilament sensitivity to Ca²⁺ and altered mechanisms of sarcoplasmic reticulum Ca²⁺ transport might partly underlie the acidosis-evoked reduction in amplitude of shortening in myocytes from control and STZ-induced diabetic rat. (Mol Cell Biochem 261: 227–233, 2004)