ABSTRACT: BACKGROUND: Brain and nervous system tumours are the most common solid cancers in children. Molecular characterisation of these tumours is important for providing novel biomarkers of disease and identifying molecular pathways which may provide putative targets for new therapies. 1H magic angle spinning NMR spectroscopy (1H HR-MAS) is a powerful tool for determining metabolite profiles from small pieces of intact tissue and could potentially provide important molecular information. METHODS: Forty tissue samples from 29 children with glial and primitive neuro-ectodermal tumours were analysed using HR-MAS (600 MHz Varian gHX nanoprobe). Tumour spectra were fitted to a library of individual metabolite spectra to provide metabolite values. These values were then used in a two tailed t-test and multi-variate analysis employing a principal component analysis and a linear discriminant analysis. Classification accuracy was estimated using a leave-one-out analysis and B632+ bootstrapping. RESULTS: Glial tumours had significantly (two tailed t-test p <0.05) higher creatine and glutamine and lower taurine, phosphoethanolamine, phosphorylcholine and choline compared with primitive neuro-ectodermal tumours. Classification accuracy was 90%. Medulloblastomas (n = 9) had significantly (two tailed t-test p <0.05) higher creatine, glutamine, phosphorylcholine, glycine and scyllo-inositol than neuroblastomas (n = 7), classification accuracy was 94%. Supratentorial primitive neuro-ectodermal tumours had metabolite profiles in keeping with other primitive neuro-ectodermal tumours whilst ependymomas (n = 2) had metabolite profiles intermediate between pilocytic astrocytomas (n = 10) and primitive neuro-ectodermal tumours. CONCLUSION: HR-MAS identified key differences in the metabolite profiles of childhood brain and nervous system improving the molecular characterisation of these tumours. Further investigation of the underlying molecular pathways is required to assess their potential as targets for new agents.