In sedentary elderly people, a reduced muscle fatty acid oxidative capacity (MFOC) may explain a decrease in whole body fat oxidation. Eleven sedentary and seven regularly exercising subjects (65.6 ± 4.5 yr) were characterized for their aerobic fitness [maximal O₂ uptake (V˙o _2 max)/kg fat free mass (FFM)] and their habitual daily physical activity level [free-living daily energy expenditure divided by sleeping metabolic rate (DEE_FLC/SMR)]. MFOC was determined by incubating homogenates of vastus lateralis muscle with [1-¹⁴C]palmitate. Whole body fat oxidation was measured by indirect calorimetry over 24 h. MFOC was 40.4 ± 14.7 and 44.3 ± 16.3 nmol palmitate · g wet tissue⁻¹ · min⁻¹ in the sedentary and regularly exercising individuals, respectively (P = nonsignificant). MFOC was positively correlated with DEE_FLC/SMR (r = 0.58, P < 0.05) but not with V˙o _2 max/kg FFM (r = 0.35, P = nonsignificant). MFOC was the main determinant of fat oxidation during all time periods including physical activity. Indeed, MFOC explained 19.7 and 30.5% of the variance in fat oxidation during walking and during the alert period, respectively (P < 0.05). Furthermore, MFOC explained 23.0% of the variance in fat oxidation over 24 h (P < 0.05). It was concluded that, in elderly people, MFOC may be influenced more by overall daily physical activity than by regular exercising. MFOC is a major determinant of whole body fat oxidation during physical activities and, consequently, over 24 h.