Small cell lung cancer (SCLC) occurs as two neuroendocrine subtypes, SCLC-C (classic) and SCLC-V (variant). One reported difference is elevated levels of diphosphodiesters (DPDE) in the more differentiated SCLC-C subtype. DPDE have been identified as primarily UDP-N-acetylhexosamines (UDP-NAH) in a variety of tumors, and changes in DPDE levels have been observed during experiments designed to induce cell differentiation. UDP-NAH synthesis is controlled by negative feedback regulation of glutamine:fructose-6-P amidotransferase (EC 2.6.1.16), which can be circumvented by glucosamine. Using 31P nuclear magnetic resonance analysis of extracts and perfused cells, we have identified UDP-N-acetylglucosamine and UDP-N-acetylgalactosamine as the primary metabolites in the DPDE spectral region of SCLC-V N-417 cells. Glucosamine addition causes a rapid increase in UDP-NAH levels. At glucosamine: glucose ratios of 1:1 and 10:1 formation of the UDP-NAH intermediates N-acetylglucosamine 6-phosphate and UDP-N-acetylglucosamine 1-phosphate is also observed, indicating UTP limitation. Subsequent uridine addition results in depletion of the intermediates and increased UDP-NAH formation. Moreover, N-417 cells retain the capacity to rapidly convert uridine to UTP despite low ATP and phosphocreatine levels. This expansion of the uridine pool may represent an additional metabolic reserve not yet addressed in the design of therapy options.