Abstract We have recently shown that recombinant thiaminase I enzyme has cytotoxic activity against breast cancer cell lines (Liu, S., Monks, N.R., Hanes, J.W., Begley, T.P., Yu, H. and Moscow, J.A. Sensitivity of breast cancer cell lines to recombinant thiaminase I. Cancer Chemotherapy and Pharmacology, 2009). Because thiamine is a cofactor in several key metabolic enzymes, including pyruvate dehydrogenase, 2-oxoglutarate dehydrogenase and transketolase, information regarding the metabolic consequences of thiaminase I treatment may increase our understanding of the cytotoxic mechanism-of-action of thiaminase I. We analyzed the metabolomes of the MCF-7 breast cancer cell line and the immortalized but non-malignant breast cell line MCF-10A with or without incubation for 4 days in medium containing thiaminase I at the predetermined cytotoxic IC50 concentrations (0.125 U/ml for MCF-7 and 0.25 U/ml for MCF-10A), with the addition of [U-13C]-labeled glucose in the final 2 days. By tracking the metabolism of [U-13C]-glucose using GCMS and NMR we were able to quantify intracellular metabolite pools and calculate both relative and absolute flux rates through several key pathways. Thiaminase I treatment decreased the amount of protein per MCF-7 cell by 30% (163 pg/cell to 115 pg/cell) while the total protein remained essentially unchanged in the MCF-10A cells (141 pg/cell vs 152 pg/cell). While both cell lines displayed increased glucose consumption and lactate production during thiaminase I treatment, these responses were greater in the MCF-7 cell line. Metabolite analysis revealed significant alterations to glycolysis and the TCA cycle in both cell lines, with glycolysis-linked metabolites being depleted in the MCF-7 cell line and changes in the equilibrium of TCA cycle intermediates in the MCF-10A cell line. The 13C- labeling patterns suggest that the MCF-10A cell line responds to thiaminase I treatment by increasing the catabolism of alternate, non-glucose carbon sources through the activation of specific metabolic pathways. In contrast, the MCF-7 cell line appears to have a less adaptable metabolic program and must down-regulate its overall metabolic throughput. Therefore, the possibility is raised that differential metabolic effects of thiaminase I could be exploited therapeutically. Supported by the Susan G. Komen For the Cure Foundation (JAM) and the California Breast Cancer Research Program (ADR). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 35.