The first aim of this study was to determine the exercise intensity that elicited the highest rate of fat oxidation in sedentary, obese subjects (OB; n=10 men, n=10 women) compared with endurance athletes (AT; n=10 men, n=10 women). The second aim was to investigate the relationship between VO₂ at the intensity eliciting the highest rate of fat oxidation and the corresponding VO₂ at the lactate threshold. Peak oxygen consumption (VO_2peak) was determined in 20 AT and 20 OB using an incremental exercise protocol on a cycle ergometer. Based on their VO_2peak values, subjects completed a protocol requiring them to exercise for 20 min at three different workloads (55, 65 and 75% VO_2peak), randomly assigned on two separate occasions. The oxidation rates of fat and carbohydrate were measured by indirect calorimetry. The highest rates of fat oxidation were at 75 % VO_2peak (AT), and at 65 % VO_2peak (OB). The rate of fat oxidation was significantly higher in AT (18.2 ± 6.1) compared with OB women (10.6 ± 4.5 kJ min^-1·kg^-1) (p < 0.01). There was no significant difference in the rate of fat oxidation for the men (AT 19.7 ± 8.1 vs. OB 17.6 ± 8.2 kJ min^-1·kg^-1). AT reached LT at a significantly (p < 0.01) higher exercise intensity expressed in VO_2peak than obese subjects (AT women 76.4 ± 0.1, men 77.3 ± 0.1 vs. OB women, 49.7 ± 0.1, men 49.5 ± 0.1% VO_2peak). A significant correlation was found between VO₂ at LT and VO₂ (L·min^-1) eliciting the maximal rate of fat oxidation in athletes (women; r = 0.67; p = 0.03; men: r = 0.75; p = 0.01) but not in the obese. In summary, we observed higher rates of fat oxidation at higher relative work rates in AT compared with OB. A significant correlation was found between LT and the exercise intensity eliciting a high rate of fat oxidation in AT (r=0.89; p < 0.01) but not in OB. Cardiorespiratory fitness, defined as VO_2peak, seems to be important in defining the relationship between a high rate of fat oxidation and LT.