Abstract Metabolic reprogramming is considered to be a hallmark of malignancy and a consequence of the mutations that cause cancer. Classical components of metabolic reprogramming include enhancements of nutrient uptake, aerobic conversion of glucose to lactate, and biosynthesis of proteins, lipids and nucleic acids. These activities are believed to collaborate to support states of supra-physiological stress resistance and proliferative capacity. However, recent work, primarily performed in cancer cell lines, has revealed an unexpected heterogeneity of metabolic programs that can support cancer cell survival and growth. It is unclear how these heterogeneous metabolic programs are regulated by the oncogenotype, and more importantly, which of the myriad activities observed in culture are relevant to bona fide tumor metabolism in vivo. I will discuss approaches to address these challenges. First, I will discuss ongoing efforts to use systematic analysis of cancer cell lines to link metabolic phenotypes with oncogenotypes, and to identify chemotherapeutic sensitivities associated with particular metabolic preferences. Second, I will describe efforts to probe metabolism in living tumors using a combination of stable isotope tracing and complementary methods. Finally, I will discuss approaches to merge metabolomics with non-invasive imaging to understand the development and consequences of metabolic heterogeneity within individual tumors. Citation Format: Ralph J. DeBerardinis. Understanding metabolic heterogeneity in cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr SY10-01. doi:10.1158/1538-7445.AM2014-SY10-01