Relationships between muscle oxidative capacity, anemia, endurance, whole-body maximal O2 consumption (VO2 max), and VO2 max work load (maximal treadmill speed at 15% grade, rat weight constant) were investigated in iron deficiency and during dietary iron repletion. Young rats were made severely iron deficient by a diet containing 2 mg iron/kg. Control animals received the same diet but with 50 mg iron/kg. Blood hemoglobin was decreased to 3.6 +/- 0.5 g/dl compared to 13.7 +/- 0.6 in control animals. The combination of decreased mitochondrial enzyme specific activities and a 30% reduction in the mitochondrial content of muscle resulted in 60-85% decreases in muscle oxidative capacities. VO2 max and VO2 max work load were both 50% lower in deficient rats, whereas endurance capacity was 90% lower in deficient animals than controls. The iron sufficient control diet was then given to deficient rats and the course of dietary repletion followed. Hemoglobin increased substantially within 3 days in parallel with VO2 max and VO2 max work load. No significant improvements in mitochondrial bioenergetic functions, mitochondrial content of muscle, muscle oxidative capacity, or endurance capacity occurred until the 5th day. We conclude that VO2 max and VO2 max work load capacity were not limited by muscle oxidative capacity. Conversely, endurance capacity was not restricted by oxygen supply, but was primarily determined by the oxidative capacity of muscle.