Abstract Tracer-based mapping of metabolic networks is a powerful approach for revealing metabolic reprogramming in human cancer. Nucleotide synthesis and turnover is critical for cell proliferation and is tightly regulated [1,2]. We have developed NMR and tandem MS methods for determining the active pathways of the synthesis of free nucleotides and nucleotide sugars including UDP-Glucose and UDPGlcNAc using multiplexed stable isotope resolved metabolomics (mSIRM)[3] in a variety of models including freshly resected lung tumor tissue, mouse PDX and organoids. Using multiple isotopic labels (13C, 2H and 15N) we determined the relative contributions to purine and pyrimidine nucleotide synthesis of both exogenous glucose, glutamine, serine and glycine and metabolically-derived intracellular precursors. We have used these approaches to compare the relative importance of different metabolic pathways in NSCLC under various conditions to assess the role of the tumor microenvironment, and how these pathways are impacted by arsenite and selenite. Our findings indicate that the substrates for de novo nucleotide synthesis may differ profoundly between cancer cell lines and fresh human lung cancer tissues; for purines, the latter preferred glucose to exogenous serine or glycine but not the former. Pyrimidine nucleotides also utilize glucose for ribose synthesis via the pentose phosphate pathway, but both glucose and glutamine for the pyrimidine rings depending on the conditions and cell type. This distinction in substrate utilization in nucleotide synthesis in human cancer tissues should be considered when targeting one-carbon metabolism for cancer therapy. Acknowledgements: This work was supported in part by grants: 1R01ES022191-01 (to TWMF and RMH), 1P01CA163223-01A1 (to ANL and TWMF), and 1U24DK097215-01A1 (to RMH, TWMF, and ANL).