Coupling of oxidation to ATP synthesis (P/O2 ratio) is a critical step in the conversion of carbon substrates to fuel (ATP) for cellular activity. The ability to quantitatively assess mitochondrial coupling in vivo can be a valuable tool for basic research and clinical purposes. At the onset of a square wave moderate exercise, the ratio between absolute amount of phosphocreatine split and O2 deficit (corrected for the amount of O2 released from the body O2 stores and in the absence of lactate production), is the mirror image of the P/O2 ratio. To calculate this value, cardiac output (Q), whole body O2 uptake (VO2), O2 deficit (O2(def)) and high-energy phosphates concentration (by 31P-NMR spectroscopy) in the calf muscles were measured on nine healthy volunteers at rest and during moderate intensity plantar flexion exercise (3.44 +/- 0.73 W per unit active muscle mass). Q and VO2 increased (from 4.68 +/- 1.56 to 5.83 +/- 1.59 l min(-1) and from 0.28 +/- 0.05 to 0.48 +/- 0.09 l min(-1), respectively), while phosphocreatine (PCr) concentration decreased significantly (22 +/- 6%) from rest to steady-state exercise. For each volunteer, "gross" O2(def) was corrected for the individual changes in the venous blood O2 stores (representing 49.9 +/- 9.5% of the gross O2(def)) yielding the "net" O2(def). Resting PCr concentration was estimated from the appropriate spectroscopy data. The so calculated P/O2 ratio amounted on average to 4.24 +/- 0.13 and was, in all nine subjects, very close to the literature values obtained directly on intact skeletal muscle. This unfolds the prospect of a non-invasive tool to quantitatively study mitochondrial coupling in vivo.