Changes in gene expression, metabolism, and energy requirements are hallmarks of cancer growth and self-sufficiency. Upregulation of the PI3K/Akt/mTor pathway in tumor cells has been shown to stimulate aerobic glycolysis, which has enabled ¹⁸F-FDG PET tumor imaging. However, of the millions of ¹⁸F-FDG PET scans conducted per year, a significant number of malignant tumors are ¹⁸F-FDG PET–negative. Recent studies suggest that several tumors may use glutamine as the key nutrient for survival. As an alternative metabolic tracer for tumors, ¹⁸F-(2S,4R)4-fluoroglutamine was developed as a PET tracer for mapping glutaminolytic tumors. A series of in vitro cell uptake and in vivo animal studies were performed to demonstrate tumor cell addiction to glutamine. Cell uptake studies of this tracer were performed in SF188 and 9L glioblastoma tumor cells. Dynamic small-animal PET studies of ¹⁸F-(2S,4R)4-fluoroglutamine were conducted in 2 animal models: xenografts produced in F344 rats by subcutaneous injection of 9L tumor cells and transgenic mice with M/tomND spontaneous mammary gland tumors. In vitro studies showed that both transformed 9L and SF188 tumor cells displayed a high rate of glutamine uptake (maximum uptake, ≈16% dose/100 μg of protein). The cell uptake of ¹⁸F-(2S,4R)4-fluoroglutamine by SF188 cells is comparable to that of ³H-l-glutamine but higher than that of ¹⁸F-FDG. The tumor cell uptake can be selectively blocked. Biodistribution and PET studies showed that ¹⁸F-(2S,4R)4-fluoroglutamine localized in tumors with a higher uptake than in surrounding muscle and liver tissues. Data suggest that certain tumor cells may use glutamine for energy production. The results support that ¹⁸F-(2S,4R)4-fluoroglutamine is selectively taken up and trapped by tumor cells. It may be useful as a novel metabolic tracer for tumor imaging.