Diabetes mellitus is a multi-component syndrome that is often complicated by angiopathy which is partly due to enhanced platelet functions. Using fluorescent dyes 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) and Fura-2 AM, changes was evaluated in the concentration of baseline and thrombin-stimulated increases in intracellular ionized calcium (Ca2+i) relative to hydrogen ions in the platelets from control, insulin-treated, and non-treated diabetic rats. The cytosol of platelets from the diabetic rats were more acidic compared to the insulin-treated and normal control rats. The increased intracellular hydrogen ion concentration [H+] or decreased pH (pH) in the diabetic rat platelets is associated with an increased baseline [Ca2+]i. Upon stimulation with thrombin, the mean peak [Ca2+]i for the insulin-treated (309 +/- 97 nmol/L) and untreated (339 +/- 135 nmol/L) diabetic rats was significantly higher than the concentration for the normal rats (213 +/- 101 nmol/L). Treatment with insulin attempts to correct the diabetes-induced elevation in the baseline of [Ca2+]i and intracellular H+. These results suggest that the relationships between Ca2+ and H+ relative to binding sites are similar in the intra- and extracellular compartments. It is our conclusion that the enhanced platelet activity and associated development of vascular diseases in diabetes may be due to an increased intracellular H+ that caused an increased baseline [Ca2+]i in diabetes mellitus.