A method for molar quantitation of in vivo proton metabolites in human brain with three-dimensional (3D) proton magnetic resonance spectroscopic imaging (MRSI) is described. The method relies on comparison of brain and calibration phantom measurements, with corrections for coil loading, and spin-lattice and spin-spin relaxation times. A 3D proton MRSI pulse sequence was developed which acquires two echoes and enables acquisition of both the TMS coil loading reference phantom and proton metabolite signals from a single experiment. With the aqueous fraction (tissue water) taken into account, the calculated molar concentrations from 24 centrum semiovale white matter voxels from 4 control subjects were (mmol/l +/- SD): N-acetyl aspartate = 14.6 +/- 2.8, total creatine+phosphocreatine = 6.0 +/- 1.2, total choline = 1.9 +/- 0.4. These values are equivalent to previously reported results obtained from single volume localized proton magnetic resonance spectroscopy.