Several factors related to the obesity can affect mitochondrial ATP biosynthesis including the uncoupling of oxidative phosphorylation (OXPHOS) by proton leak induced by accumulated free fatty acids (FFA), the over-expression of uncoupling proteins (UCPs) and altered expression of ATPase inhibitory protein factors 1 (IF1). Therefore, the present work was aimed to evaluate the activity of ATP synthase in isolated mitochondria from skeletal and heart muscle of animal models of obesity induced by sucrose diet at different periods. Short periods of sucrose intake (6 and 12 weeks) are sufficient to induce fat accumulation, hypertriglyceridemia and high plasma FFA. However, a significant decline in the activity of the ATP synthase starts to be obvious in mitochondria from skeletal muscle after 24 weeks of sucrose consumption. This impairment of ATP synthase is associated with increased FFA in skeletal muscle homogenate. Both skeletal and heart muscle ATP synthase activity were found sensitive to oleic acid and to GDP, a physiological inhibitor of UCPs, found enhanced by aging. In addition, sucrose diet increases the IF1 content in both skeletal and heart muscle probably to avoid the hydrolysis activity of ATP synthase. In mitochondria from heart muscle a decrease in the ATP synthase activity was only observed according to the age in both groups of rats and it was not affected by sucrose feeding. Our results suggest that the decline of the ATP synthase activity in mitochondria from skeletal muscle can be due to the accumulation of FFA in skeletal muscle tissue as uncouplers, and the IF1 over-expression induced by sucrose diet is an adaptative mechanism to inhibit the ATP synthase hydrolysis activity.