Abstract Both sleep and glutamatergic signaling in the brain are tightly controlled and homeostatically regulated. Sleep homeostasis is reliably reflected by predictable changes in brain electrical activity in waking and sleep, yet the underlying molecular mechanisms remain elusive. Current hypotheses posit that recovery sleep following prolonged waking restores efficient functioning of the brain, for example by keeping glutamatergic signaling in a homeostatic range. We recently provided evidence in humans and mice that metabotropic glutamate receptors of subtype-5 (mGluR5) contribute to the brain’s coping mechanisms with sleep deprivation. Here we combined in 31 healthy men, proton magnetic resonance spectroscopy to measure the levels of glutamate (Glu), GLX (glutamate-to-glutamine ratio) and GABA (γ-amino-butyric-acid) in basal ganglia (BG) and dorsolateral prefrontal cortex, simultaneous positron emission tomography to quantify mGluR5 availability with the novel radioligand, [ 18 F]PSS232, and quantification in blood plasma of the mGluR5-regulated proteins, fragile-X mental retardation protein (FMRP) and brain-derived neurotrophic factor (BDNF). All measurements were conducted at the same circadian time in baseline, following sleep deprivation and after recovery sleep. We found that Glu and GLX in BG (p all < 0.01), but not in prefrontal cortex, and the plasma concentration of FMRP (p < 0.02), were increased after sleep loss and tended to normalize following recovery sleep (p all < 0.1). Furthermore, a night without sleep enhanced whole-brain and striatal mGluR5 availability and was normalized by recovery sleep (p all < 0.05). By contrast, other brain metabolites and plasma BDNF levels were not altered. The findings demonstrate convergent changes in distinct markers of glutamatergic signaling across prolonged wakefulness and recovery sleep in humans. They warrant further studies to elucidate the underlying mechanisms that link the homeostatic regulation of sleep and glutamatergic system activity in health and disease. One-sentence summary Sleep-dependent recovery of wakefulness-induced changes in, cerebral glutamatergic signaling Major subject area Neuroscience; Human Biology & Medicine