We used 31-phosphorus magnetic resonance spectroscopy to study in vivo the transport of inorganic phosphate (Pi) from cytosol into mitochondria as assessed by the kinetics of Pi recovery during ischemia after aerobic exercise in human skeletal muscle. In all subjects during the first 30 s of ischemia inorganic phosphate showed a marked decrease from the value measured at the end of exercise, whilst phosphocreatine maintained the same value reached at the end of exercise. Our results show that Pi transport from cytosol into mitochondria is active in the absence of ATP biosynthesis and lasts 30 s possibly as a consequence of a decreased pH gradient, due to symport of Pi and H+ associated with an inactive electron transport chain during ischemia.