The electrochemical gradient of protons, delta mu H+, was estimated in the obligatory aerobic yeast Rhodotorula glutinis in the pH0 range from 3 to 8.5. The membrane potential, delta psi, was measured by steady-state distribution of the hydrophobic ions, tetraphenylphosphonium (TPP+) for negative delta psi above pH0 4.5, and thiocyanate (SCN-) for positive delta psi below pH0 4.5. The chemical gradient of H+ was determined by measuring the chemical shift of intracellular Pi by 31P-NMR at given pH0 values. The values of pHi increased almost linearly from 7.3 at pH0 3 to 7.8 at pH0 8.5. In the physiological pH0 range from 3.5 to 6, delta mu H+ was fairly constant at values between 17-18 KJ mol-1, gradually decreasing at pH0 above 6. In deenergized cells, the intracellular pHi decreased to values as low as 6, regardless of whether the cell suspension was buffered at pH0 4.5 or 7.5. There was no membrane potential detectable in deenergized cells.