The effect of docosahexaenoic acid (DHA) intake on cardiac mitochondrial function was evaluated in permeabilized fibers in insulin deficiency and insulin-resistance in rats. The insulin deficient state was obtained by streptozotocin injection 2 months before investigations. Insulin resistance was obtained by feeding a 62% fructose diet for three months. DHA was incorporated in the diet to modify the fatty acid composition of cardiac membranes, including mitochondria. Insulin deficiency decreased mitochondrial creatine kinase (mi-CK) activity and mitochondrial sensitivity to ADP. DHA intake prevented these alterations. Moreover, the insulin deficient state significantly decreased n-3 polyunsaturated fatty acids (PUFA) and slightly increased n-6 PUFA in both cardiac and mitochondrial membranes, inducing a significant increase in n-6/n-3 ratio. DHA intake maintained high myocardial and mitochondrial DHA content. Insulin deficiency also decreased glutamate and palmitoylcarnitine-supported mitochondrial respiration, but DHA intake did not prevent these effects. In contrast, insulin resistance did not affect mi-CK activity or sensitivity to ADP. However, insulin resistance influenced the myocardial fatty acid composition with decreased n-6 and n-3 PUFA contents and increased monounsaturated fatty acid content. Only slight alterations were observed in mitochondrial fatty acid composition and they were corrected by DHA intake. Moreover, insulin resistance decreased the glutamate-supported respiration, and DHA intake did not influence this effect. In conclusion, the impairment of cardiac mitochondrial function was more pronounced in insulin deficient state than in insulin resistance. The modification of fatty acid composition of cardiac and mitochondrial membranes by DHA partially prevented the mitochondrial alterations induced in the two models.