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The effect of temperature on31P NMR spectra from isolated perfused rat livers was studied at 9.4 T. Relaxation times (T1andT2) of nucleoside triphosphates (NTP) and inorganic phosphate (Pi) were determined at 37, 25, 15, and 4°C. Under hypothermic conditions, an unexpected apparent line sharpening in the Pi spectral region and a clear emergence of an additional Pi resonance were observed. This additional signal was assigned to mitochondrial Pi.T1values obtained for cytosolic and mitochondrial Pi at 4°C were 1.14 ± 0.24 s (n= 5) and 0.71 ± 0.18 s (n= 5), respectively. No significant mitochondrial contribution to the Pi resonance was observed at 37°C. Quantification of Pi and NTP liver contents at 37 and 4°C was performed by comparing the perfused liver spectrum and the corresponding perchloric acid extract spectrum. Under experimental conditions of low external Pi (0.12 mM), it was concluded that intracellular Pi was completely NMR-visible at 4 and 37°C. The observation of the mitochondrial Pi signal at 4°C was well explained by an increase in the Pi level within the matrix, in response to the mitochondrial swelling induced by hypothermia, as observed by electron microscopy.T2values for the cytosolic Pi at 37 and 4°C were 17 ± 4 ms (n= 8) and 22 ± 4 ms (n= 10), respectively. Comparison with measured linewidths indicated that line broadening for the main phosphorylated metabolites—including matrix Pi—was the result ofB0field inhomogeneity. The additional broadening of the cytosolic Pi resonance at 4 and 37°C was attributed to pH heterogeneity within the liver.