Early and late effects of alloxan diabetes and insulin treatment on mitochondrial membrane structure and function were evaluated by studying the kinetic properties of mitochondrial membrane marker enzyme FoF₁-ATPase and its modulation by membrane lipid/phospholipid composition and membrane fluidity. Under all experimental conditions the enzyme displayed three kinetically distinguishable components. In 1wk-old diabetic animals the enzyme activity was unchanged; however, K _m and V _max of component I increased and K _m of component II decreased. Insulin treatment resulted in lowering of K _m and V _max of components II and III. One-mon diabetic state resulted in decreased enzyme activity, whereas insulin treatment caused hyperstimulation. K _m of components I and II decreased together with decreased V _max of all the components. Insulin treatment restored the K _m and V _max values. In late-stage diabetes the catalytic efficiency of components I and II increased; insulin treatment had drastic adverse effect. Binding pattern of ATP was unchanged under all experimental conditions. Diabetic state resulted in progressive decrease in energy of activation in the low temperature range (E _L). Insulin treatment lowered the energy of activation in the high temperature range (E _H) without correcting the E _L values. The phase transition temperatures increased in diabetic state and were not corrected by insulin treatment. Long-term diabetes lowered the total phospholipid content and elevated the cholesterol content; insulin treatment had partial restorative effect. The membrane fluidity decreased in general in diabetic condition and was not corrected by insulin treatment at late stage. Regression analysis studies suggest that specific phospholipid classes and/or their ratios may play a role in modulation of the enzyme activity.