Intact mitochondria were studied by high-resolution 31P nuclear magnetic resonance. Observable internal phosphate compounds included inorganic phosphate (Pi), ADP, and ATP. The internal pH was determined by the chemical shift of the internal Pi, the pK2 (6.7) of which was measured in uncoupled mitochondria. The observed equilibrium relation between the internal and the external Pi was consistent with the exchange equilibrium through the H2PO4-/OH- carrier. The internal ATP and ADP were essentially Mg2+ bound and their resonances were distinguishable from those of the external ATP and ADP by the chemical shift differences due to the Mg2+ concentration gradient and deltapH. Oxidative phosphorylation was followed by the separate resonances of Pi and adenine nucleotides both internal and external to mitochondria. From these resonances the internal and external phosphate potentials could be estimated.