Magnetic resonance (MR) spectroscopy provides biochemical information about tumors. The authors sought to determine the relationship between in vivo and ex vivo biochemical characterization of pediatric brain tumors by using MR spectroscopy. Their hypothesis was that ex vivo MR spectroscopy provides a link between in vivo MR spectroscopy and neuropathological analysis. In vivo proton MR spectroscopy was performed before surgery in 11 patients with neuroepithelial tumors. During resection, a total of 40 tumor biopsy samples were obtained from within the volume of interest identified on in vivo MR spectroscopy and were frozen immediately in liquid nitrogen. High-Resolution Magic Angle Spinning (HRMAS) was used to perform ex vivo MR spectroscopy in these 40 tumor biopsy samples. Neuropathological analysis was performed using the same biopsy samples, and the tumors were classified as ependymoma, choroid plexus carcinoma, pineoblastoma (one each), and pilocytic astrocytoma, medullobastoma, low-grade glioma, and glioblastoma multiforme (two each). Ex vivo HRMAS MR spectroscopy improved line widths and line shapes in the spectra, compared with in vivo MR spectroscopy. Choline (Cho) detected in vivo corresponded to three different peaks ex vivo (glycerophosphocholine, phosphocholine PCho, and Cho). Metabolite ratios from in vivo spectra correlated with ratios from ex vivo spectra (Pearson correlation coefficient range r = 0.72-0.91; p < or = 0.01). Metabolite ratios from ex vivo spectra, such as PCho/ total creatine (tCr) and lipid/tCr, correlated with the percentage of cancerous tissue and percentage of tumor necrosis, respectively (r = 0.84; p < 0.001). Agreement between in vivo and ex vivo MR spectroscopy indicates that ex vivo HRMAS MR spectroscopy can improve resolution of this modality and provide a link between in vivo MR spectroscopy and neuropathological analysis.