Oxygen (O(2)) extraction is impaired in exercising skeletal muscle of humans with mutations of mitochondrial DNA (mtDNA), but the muscle hemodynamic response to exercise has never been directly investigated. This study sought to examine the extent to which human skeletal muscle perfusion can increase without reductions in blood oxygenation and to determine whether erythrocyte O(2) off-loading and related ATP vascular mechanisms are impaired in humans with mutations of mtDNA. Leg vascular hemodynamic, oxygenation and ATP were investigated in ten patients with mtDNA mutations and ten matched healthy control subjects: 1) at rest during normoxia, hypoxia, hyperoxia and intra-femoral artery ATP infusion, and 2) during passive and dynamic one-legged knee-extensor exercises. At rest, blood flow (LBF), femoral arterial and venous blood oxygenation and plasma ATP were similar in the two groups. During dynamic exercise, LBF and vascular conductance increased 9-10 fold in the patients despite erythrocyte oxygenation and leg O(2) extraction remained unchanged (p