Phosphatidylethanolamine N-methylation was examined in cardiac subcellular membranes after inducing chronic experimental diabetes in rats (65 mg streptozotocin/kg, i.v.). The incorporation of radiolabeled methyl groups from S-adenosyl-L-methionine in diabetic sarcolemma was significantly depressed at all three catalytic sites (I, II, and III) of the methyltransferase system. An increase in methyl group incorporation was evident at site I without any changes at sites II and III in diabetic sarcoplasmic reticulum and mitochondria. Similar changes were also seen for the individual N-methylated lipids (monomethyl-, dimethylphosphatidylethanolamine, and phosphatidylcholine) specifically formed at each catalytic site in all cardiac membranes from diabetic animals. These alterations in N-methylation were reversible by a 14-d insulin therapy to the diabetic animals. In the presence of 10 microM ATP and 0.1 microM Ca2+, N-methylation was maximally activated at site I in both control and diabetic sarcolemma and sarcoplasmic reticulum, but not in mitochondria. Incubation of cardiac membranes with of S-adenosyl-L-methionine showed that Ca2(+)-stimulated ATPase activities in both sarcolemma and sarcoplasmic reticulum were augmented; however, the activation of diabetic sarcolemma was lesser and that of diabetic sarcoplasmic reticulum was greater in comparison with the control preparations. These results identify alterations in phosphatidylethanolamine N-methylation in subcellular membranes from diabetic heart, and it is suggested that these defects may be crucial in the development of cardiac dysfunction in chronic diabetes.