Quantitation of metabolite concentrations by NMR spectroscopy is complicated by the need to determine the volume from which signals are detected, and by the need to obtain the relative sensitivity of detection within this volume. The use of coils with inhomogeneous B1 fields further complicates these problems. In order to quantify metabolite concentrations using 31P NMR spectroscopy, an external reference of hexamethyl phosphoroustriamide was used. Studies were performed on phantoms, using either a surface coil or a Helmholtz head coil to confirm the accuracy of both the ISIS volume selection technique and the use of an external reference. The limitations of this method are related to contamination and signal loss inherent in the ISIS technique and difficulties with integration of broad overlapping peaks. The method was applied to seven normal human subjects. The integrals for metabolite signals in normal brain and calf muscle were determined by using NMRI software. The T1 values of the signals of all phosphorus metabolites in the selected volume were measured in order to correct for saturation effects. The concentrations for PCr, Pi, and ATP were 4.9, 2.0, and 2.5 mM in brain and 36.5, 5.7, and 7.3 mM in muscle. These results are in good agreement with those reported for animals, demonstrating the validity of this quantitation technique.