Patterns of fuel use during locomotion are determined by exercise intensity and duration, and are remarkably similar across many mammalian taxa. However, as lipids have a high yield of ATP per mole and are stored in large quantities, their use should be favored in endurance-adapted animals. To examine the capacity for alteration or differential regulation of fuel-use patterns, we studied two lines of mice that had been selectively bred for high voluntary wheel running (HR), including one characterized by small hindlimb muscles (HR(mini)) and one without this phenotype (HR(normal)), as well as a nonselected control line. We evaluated: 1) maximal aerobic capacity (Vo(2 max)); 2) whole body fuel use during exercise by indirect calorimetry; 3) cardiac properties; and 4) many factors involved in regulating lipid use. HR mice achieved an increased Vo(2 max) compared with control mice, potentially in part due to HR cardiac capacities for metabolic fuel oxidation and the larger relative heart size of HR(mini) mice. HR mice also exhibited enhanced whole body lipid oxidation rates at 66% Vo(2 max), but HR(mini), HR(normal), and control mice did not differ in the proportional mix of fuels sustaining exercise (% total Vo(2)). However, HR(mini) gastrocnemius muscle had elevated fatty acid translocase (FAT/CD36) sarcolemmal protein and cellular mRNA, fatty acid binding protein (H-FABP) cytosolic protein, peroxisome proliferator-activated receptor (PPAR) α mRNA, and mass-specific activities of citrate synthase, β-hydroxyacyl-CoA dehydrogenase, and hexokinase. Therefore, high-running mouse lines had whole body fuel oxidation rates commensurate with maximal aerobic capacity, despite notable differences in skeletal muscle metabolic phenotypes.