Sixteen metabolites were quantified from 11–24 μl volumes in three different brain regions (hippocampus, striatum, and cerebral cortex) during postnatal development. Rat pups from the same litter were repeatedly measured on postnatal days 7, 10, 14, 21, and 28 using a completely noninvasive and longitudinal study design. Metabolite quantification was based on ultra-short echo-time 1H NMR spectroscopy at 9.4 T and LCModel processing. Most of the brain metabolites were quantified with Cramer-Rao lower bounds (CRLB) less than 20%, which corresponded to an estimated concentration error <0.2 μmol/g. Taurine and total creatine were quantified with CRLB ≤ 5% from all 114 processed spectra. The resulting high reliability and reproducibility revealed significant regional and age-related changes in metabolite concentrations. The most sensitive markers for developmental and regional variations between hippocampus, striatum, and cerebral cortex were N-acetylaspartate, myo-inositol, taurine, glutamate, and choline compounds. Absolute values of metabolite concentrations were in very good agreement with previously published in vitro results based on chromatographic measurements of brain extracts. The current data may serve as a reference for studies focused on developmental defects and pathologies using neonatal rat models. Magn Reson Med 50:24–32, 2003. © 2003 Wiley-Liss, Inc.