The energy metabolism and the brain intracellular pH regulation under arterial CO2 tensions of 25-90 mm Hg were investigated in unanesthetized spontaneously breathing rats by in vivo phosphorus nuclear magnetic resonance spectroscopy (31P NMR). The 31P brain spectra, recorded with a high resolution spectrometer (AM 400 Brucker), allowed repeated non-invasive measurements of cerebral pH (pHi), phosphocreatine (PCr), inorganic phosphate (Pi) and adenosine triphosphate (ATP) levels in 15 rats breathing a gas mixture containing 21% O2, N2, and a varied percentage of CO2. The pHi decreased significantly when the paCO2 was increased by hypercapnia. The percentage of pH regulation, estimated from the linear regression analysis of pHi versus the logarithm of the paCO2 was 78%. This result indicates that spontaneously breathing unanesthetized animals have better pHi regulation under hypercapnia investigated than that estimated for higher levels of hypercapnia in previous studies on unanesthetized animals, suggesting that there is a threshold for this highly efficient regulation. Furthermore, there were no significant correlations between the PCr, ATP and Pi levels and the paCO2 levels during hypercapnia. This indicates that physiological variations of the CO2 tension in the blood, and consequently in the brain parenchyma, have little effect on cerebral energy metabolism in unanesthetized spontaneously breathing animals.