Glucose and glutamine are the two principal nutrients that cancer cells use to proliferate and survive. Many cancers show altered glucose metabolism, which constitutes the basis for in vivo positron emission tomography (PET) imaging with (18)F-fluorodeoxyglucose ((18)F-FDG). However, (18)F-FDG is ineffective in evaluating gliomas because of high background uptake in the brain. Glutamine metabolism is also altered in many cancers, and we demonstrate that PET imaging in vivo with the glutamine analog 4-(18)F-(2S,4R)-fluoroglutamine ((18)F-FGln) shows high uptake in gliomas but low background brain uptake, facilitating clear tumor delineation. Chemo/radiation therapy reduced (18)F-FGln tumor avidity, corresponding with decreased tumor burden. (18)F-FGln uptake was not observed in animals with a permeable blood-brain barrier or neuroinflammation. We translated these findings to human subjects, where (18)F-FGln showed high tumor/background ratios with minimal uptake in the surrounding brain in human glioma patients with progressive disease. These data suggest that (18)F-FGln is avidly taken up by gliomas, can be used to assess metabolic nutrient uptake in gliomas in vivo, and may serve as a valuable tool in the clinical management of gliomas.