The effect of an increase in dietary fat content on fat and carbohydrate balances and energy expenditure (EE) was studied in nine formerly obese women with genetic predisposition to obesity (postobese) and a closely matched control group. Isocaloric low- (20% fat energy) and high-fat diets (50%) were consumed for 3 days preceding and during a 24-h respiratory chamber stay, whereas a medium-fat diet (30%) was consumed only on the day of measurement. After adjustment for 24-h energy intake to equal 24-h EE, 24-h fat balance was increased when the dietary fat content increased (P < 0.0002). No differences in macronutrient balances were found on the low-fat and medium-fat diets, but on the high-fat diet the postobese women failed to increase ratio of fat to carbohydrate oxidation appropriately (0.59 g/g, 95% confidence interval 0.47-0.67 vs. controls 1.02 g/g, 0.88–1.12; P = 0.002). This caused a positive adjusted fat balance (+11.0 g/day, 2.3–19.6 vs. controls -8.9 g/day, -17.5 to -0.2; P < 0.001) and a negative carbohydrate balance (-41.8 g/day, -69.5 to -14.0 vs. controls +23.2 g/day, -4.6 to +50.9; P < 0.001). Decreasing the dietary fat content increased 24-h EE in the postobese women (P = 0.02), whereas it was unaffected in the control group. Independent of energy balance, an increase in dietary fat content to 50% fat energy results in preferential fat storage, impaired suppression of carbohydrate oxidation, and reduction of 24-h EE in postobese women.