To determine the sequence of alterations in cardiac sarcolemmal (SL) Na+-Ca2+ exchange, Na+-K+ ATPase and Ca2+-transport activities during the development of diabetes, rats were made diabetic by an intravenous injection of 65 mg/kg alloxan. SL membranes were prepared from control and experimental hearts 1-12 weeks after induction of diabetes. A separate group of 4 week diabetic animals were injected with insulin (3 U/day) for an additional 4 weeks. Both Na+-K+ ATPase and Ca2+-stimulated ATPase activities were depressed as early as 10 days after alloxan administration; Mg2+ ATPase activity was not depressed throughout the experimental periods. Both Na+-Ca2+ exchange and ATP-dependent Ca2+-uptake activities were depressed in diabetic hearts 2 weeks after diabetes induction. These defects in SL Na+-K+ ATPase and Ca-transport activities were normalized upon treatment of diabetic animals with insulin. Northern blot analysis was employed to compare the relative mRNA abundances of α1-subunit of Na+-K+ ATPase and Na+-Ca2+ exchanger in diabetic ventricular tissue vs. control samples. At 6 weeks after alloxan administration, a significant depression of the Na+-K+ ATPase α1- subunit mRNA was noted in diabetic heart. A significant increase in the Na+-Ca2+ exchanger mRNA abundance was observed at 3 weeks which returned to control by 5 weeks. The results from the alloxan-rat model of diabetes support the view that SL membrane abnormalities in Na+-K+ ATPase, Na+Ca2+ exchange and Ca2+-pump activities may lead to the occurrence of intracellular Ca2+overload during the development of diabetic cardiomyopathy but these defects may not be the consequence of depressed expression of genes specific for those SL proteins.