Previous 31P nuclear magnetic resonance (NMR) studies have measured the concentrations of phosphates, free Mg2+, pH and flux through enzyme-catalyzed reactions in a variety of tissues. A surgically-implanted coil has been developed to measure these parameters in the rat liver in vivo, and to assess the effect of external perturbations on the concentrations and physiological environment of phosphorus metabolities in the liver. The sensitive volume and optimal pulse were determined for the coil, which was insulated to exclude signal from surrounding tissues. The metabolic stability of the liver during acquisition of spectra was demonstrated by normal values for [Pi], [ATP], [lactate], and [pyruvate] in livers which were freeze-clamped immediately after completion of the NMR experiment. The stability was also confirmed by constant values for intracellular pH (7.2), free [Mg2+] (0.7 mM), and NMR detectable [Pi]/[ATP]. The sensitivity of the 31P-NMR spectrum of the liver in vivo to the physiological state of the animals was illustrated by comparing spectra from fed and 48 h fasted rats. The major qualitative differences were an increase in the pyridine nucleotide/adenine nucleotide ratio, and a small, but consistent shift in the frequency of the composite phosphomonoester peak. The spin-lattice relaxation time of each major phosphate resonance was measured in vivo using a modified homospoil saturation recovery pulse sequence; the T1 of ATP gamma-phosphate was 0.17 s. Selective saturation experiments did not detect magnetization transfer between the ATP gamma-phosphate and inorganic phosphate.