Changes in skeletal muscle respiratory capacity parallel that of aerobic fitness. It is unknown whether mitochondrial content, alone, can fully account for these differences in skeletal muscle respiratory capacity. The aim of the present study was to examine quantitative and qualitative mitochondrial characteristics across four different groups (n = 6 each), separated by cardiorespiratory fitness. High-resolution respirometry was performed on muscle samples to compare respiratory capacity and efficiency in active, well-trained, highly trained, and elite individuals. Maximal exercise capacity (ml O(2)·min(-1)·kg(-1)) differed across all groups, with mean ± SD values of 51 ± 4, 64 ± 5, 71 ± 2, and 77 ± 3, respectively. Mitochondrial content assessed by citrate synthase activity was higher in elite trained compared with active and well-trained (29 ± 7 vs. 16 ± 4 and 19 ± 4 nmol·min(-1)·mg wet wt(-1), respectively). When normalizing respiration to mitochondrial content, the respiratory capacities during maximal fatty acid oxidation (P = 0.003), maximal state 3 respiration (P = 0.021), and total electron transport system capacity (P = 0.008) improved with respect to maximal exercise capacity. The coupling efficiency of β-oxidation, however, expressed no difference across groups. These data demonstrate the quantitative and qualitative differences that exist in skeletal muscle mitochondrial respiratory capacity and efficiency across individuals that differ in aerobic capacity. Mitochondrial-specific respiration capacities during β-oxidation, maximal oxidative phosphorylation, and electron transport system capacity all correspondingly improve with aerobic capacity, independent of mitochondrial content in human skeletal muscle.