Liver dysfunction correlates with alterations of intracellular concentrations of 31P metabolites. Localization and absolute quantification should help to trace regional hepatic metabolism. An improved protocol for the absolute quantification of 31P metabolites in vivo in human liver was developed by employing three-dimensional (3D) k-space weighted spectroscopic imaging (MRSI) with B1-insensitive adiabatic excitation. The protocol allowed for high spatial resolution of 17.8 ± 0.22 cm3 in 34 min at 3 T. No pulse adjustment prior to MRSI measurement was necessary due to adiabatic excitation. The protocol geometry was identical for all measurements so that one calibration data set, acquired from phantom replacement measurement, was applied for all quantifications. The protocol was tested in 10 young, healthy volunteers, for whom 57 ± 7 spectra were quantified. Concentrations per liter of liver volume (reproducibilities) were 2.24 ± 0.10 mmol/L (1.8%) for phosphomonoesters (PME), 1.37 ± 0.07 mmol/L (7.9%) for inorganic phosphate (Pi), 11.40 ± 0.96 mmol/L (2.9%) for phosphodiesters (PDE), and 2.14 ± 0.10 mmol/L (1.6%) for adenosine triphosphate (ATP), respectively. Taken together, this approach provides fast, simple, and reproducible high-resolution absolute quantification and detailed mapping of the spatial distribution of hepatic 31P metabolites. This method allows for examination of regional deviations of energy metabolism in human liver diseases. Magn Reson Med 60:796–802, 2008. © 2008 Wiley-Liss, Inc.