Neuroimaging methods have considerably developed over the last decades and offer various noninvasive approaches for measuring cerebral metabolic fluxes connected to energy metabolism, including PET and magnetic resonance spectroscopy (MRS). Among these methods, 31 P MRS has the particularity and advantage to directly measure cerebral ATP synthesis without injection of labeled precursor. However, this approach is methodologically challenging, and further validation studies are required to establish 31 P MRS as a robust method to measure brain energy synthesis. In the present study, we performed a multimodal imaging study based on the combination of 3 neuroimaging techniques, which allowed us to obtain an integrated picture of brain energy metabolism and, at the same time, to validate the saturation transfer 31 P MRS method as a quantitative measurement of brain ATP synthesis. A total of 29 imaging sessions were conducted to measure glucose consumption (CMRglc), TCA cycle flux (VTCA), and the rate of ATP synthesis (VATP) in primate monkeys by using 18 F-FDG PET scan, indirect 13 C MRS, and saturation transfer 31 P MRS, respectively. These 3 complementary measurements were performed within the exact same area of the brain under identical physiological conditions, leading to: CMRglc ؍ 0.27 ؎ 0.07 mol⅐g ؊1 ⅐min ؊1 , VTCA ؍ 0.63 ؎ 0.12 mol⅐g ؊1 ⅐min ؊1 , and VATP ؍ 7.8 ؎ 2.3 mol⅐g ؊1 ⅐min ؊1 . The consistency of these 3 fluxes with literature and, more interestingly, one with each other, demonstrates the robustness of saturation transfer 31 P MRS for directly evaluating ATP synthesis in the living brain.glycolysis ͉ TCA cycle ͉ oxidative phosphorylation ͉ NMR spectroscopy ͉ metabolic fluxes N umerous brain disorders, like neurodegenerative diseases, are associated with impairment in energy metabolism. This observation has been driving considerable technological developments in medical imaging, aiming at measuring brain energy metabolism. PET combined with 18 F-2-f luoro-2-deoxy-Dglucose ( 18 F-FDG) injection has been used in research on normal and pathological brain for Ϸ30 years (1-3). More recently, magnetic resonance spectroscopy (MRS) has brought new tools for imaging cerebral energy fluxes (4-16).However, methodological developments are still needed to answer the clinical need for earlier diagnosis and follow-up of neurodegenerative pathologies. Although PET detection of 18 F-FDG has proven efficient to map cerebral glucose consumption (CMRglc), this technique does not directly reflect energy storage and utilization that is mainly derived from glucose oxidation. Also, PET is unlikely to become widely accessible, due to its invasiveness and cost. However, magnetic resonance is of widespread use for anatomical imaging, and clinical scanners can be equipped for metabolic imaging at limited cost. Indeed, MRS has proven powerful to measure brain energy metabolism by detecting 13 C, 17 O, or 31 P nuclei, which only require dedicated radiofrequency components. Quantitative measurement of cerebral oxidative metabol...