Altered glutamate signaling is thought to be involved in a myriad of psychiatric disorders. Positron emission tomography (PET) imaging with [18F]FPEB allows assessing dynamic changes in metabotropic glutamate receptor 5 (mGluR5) availability underlying neuropathological conditions. The influence of endogenous glutamatergic levels into receptor binding has not been well established yet. The purpose of this study was to explore the [18F]FPEB binding regarding to physiological fluctuations or acute changes of glutamate synaptic concentrations by a translational approach; a PET/MRS imaging study in 12 healthy human volunteers combined to a PET imaging after an N-acetylcysteine (NAc) pharmacological challenge in rodents. No significant differences were observed with small-animal PET in the test and retest conditions on the one hand and the NAc condition on the other hand for any regions. To test for an interaction of mGuR5 density and glutamatergic concentrations in healthy subjects, we correlated the [18F]FPEB BPND with Glu/Cr, Gln/Cr, Glx/Cr ratios in the anterior cingulate cortex VOI; respectively, no significance correlation has been revealed (Glu/Cr: r = 0.51, p = 0.09; Gln/Cr: r = -0.46, p = 0.13; Glx/Cr: r = -0.035, p = 0.92).These data suggest that the in vivo binding of [18F]FPEB to an allosteric site of the mGluR5 is not modulated by endogenous glutamate in vivo. Thus, [18F]FPEB appears unable to measure acute fluctuations in endogenous levels of glutamate.