Abstract Somatic hotspot mutations in the NADP+-dependent isocitrate dehydrogenases, IDH1 and IDH2, arise frequently in gliomas. These gliomas include WHO grades II and III oligodendrogliomas and astrocytomas, as well as WHO grade IV secondary glioblastomas. When mutated, IDH1 and IDH2 gain the ability to produce (R)-2-hydroxyglutarate (2HG) and may dominant-negatively inhibit IDH1-WT. Aside from these specific functions, the downstream effect of these metabolic enzyme mutations on cellular metabolism is unknown. To identify metabolic alterations caused by IDH1 and IDH2 mutants, we used three mass spectrometry methods (GC-MS/MS, LC-MS/MS +ESI, and LC-MS/MS -ESI) to profile >200 biochemicals in lysates of human oligodendroglial cell line (HOG) cells that homologously express IDH1-R132H or IDH2-R172K, which are the most frequent IDH1 and IDH2 mutants in glioma, respectively. To determine whether 2HG production or inhibition of IDH1-WT could mediate similar effects on cellular metabolism as IDH mutant expression, we also analyzed cells treated cells with either 7.5mM or 30mM 2HG as well as cells with stable shRNA knockdown of endogenous IDH1-WT. Unsupervised hierarchical clustering, univariate threshold tests, correlation analysis, and principal component analysis revealed that cells expressing IDH1-R132H and IDH2-R172K have similar metabolite profiles, and that 2HG treatment also results in a similar profile. However, cell with shRNA IDH1 knockdown shared few metabolic features with cells expressing IDH mutants. Metabolite set enrichment analyses revealed that numerous amino acids, N-acetylated amino acids, TCA cycle metabolites, glutathione metabolites, and choline derivatives were markedly altered in IDH-mutant expressing cells. Strikingly, we found that N-acetyl-aspartyl-glutamate (NAAG), a common brain metabolite, is 50-fold reduced in cells expressing IDH1-R132H and >2-fold reduced in human glioma tissues with IDH1-R132H compared to glioma tissues without IDH mutations. This work shows that IDH mutants can induce widespread cellular metabolic changes. Additionally, the metabolites that were found to be altered in this study may play a role in glioma pathogenesis and may be useful for diagnosis or treatment for gliomas. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-257. doi:10.1158/1538-7445.AM2011-LB-257