BACKGROUND: Obesity may be associated with a lowered use of fat as a fuel, which may contribute to the enlarged adipose tissue stores. AIM: The aim of the present study was to study fatty acid use in the fasting state and in response to a high fat load in a large cohort of obese subjects (n = 701) and a lean reference group (n = 113). METHODS: Subjects from eight European centers underwent a test meal challenge containing 95 en% fat [energy content 50% of estimated resting energy expenditure (EE)]. Fasting and postprandial fat oxidation and circulating metabolites and hormones were determined over a 3-h period. RESULTS: Postprandial fat oxidation (as percent of postprandial EE, adjusted for fat mass, age, gender, center, and energy content of the meal) decreased with increasing body mass index (BMI) category (P < 0.01), an effect present only in those obese subjects with a relatively low fasting fat oxidation (below median, interaction BMI category x fasting fat oxidation, P < 0.001). Fasting fat oxidation increased with increasing BMI category (P < 0.001), which was normalized after adjustment for fat-free mass and fat mass. Furthermore, insulin resistance was positively associated with postprandial fat oxidation (P < 0.05) and negatively associated with fasting fat oxidation (expressed as percent of EE), independent of body composition. CONCLUSIONS: The present data indicate an impaired capacity to regulate fat oxidation in the obese insulin-resistant state, which is hypothesized to play a role in the etiology of both obesity and insulin resistance.