The adult brain is a high-glucose and oxygen-dependent organ, with an extremely organized network of cells and large energyconsuming synapses. To reach this level of organization, early stages in development must include an efficient control of cellular events and regulation of intracellular signaling molecules and ions such as hydrogen peroxide (H 2 O 2 ) and calcium (Ca 2+ ), but in cerebral tissue, these mechanisms of regulation are still poorly understood. Hexokinase (HK) is the first enzyme in the metabolism of glucose and, when bound to mitochondria (mtHK), it has been proposed to have a role in modulation of mitochondrial H 2 O 2 generation and Ca 2+ handling. Here, we have investigated how mtHK modulates these signals in the mitochondrial context during postnatal development of the mouse brain. Using high-resolution respirometry, western blot analysis, spectrometry and resorufin, and Calcium Green fluorescence assays with brain mitochondria purified postnatally from day 1 to day 60, we demonstrate that brain HK increases its coupling to mitochondria and to oxidative phosphorylation to induce a cycle of ADP entry/ATP exit of the mitochondrial matrix that leads to efficient control over H 2 O 2 generation and Ca 2+ uptake during development until reaching plateau at day 21. This contrasts sharply with the antioxidant enzymes, which do not increase as mitochondrial H 2 O 2 generation escalates. These results suggest that, as its use of glucose increases, the brain couples HK to mitochondria to improve glucose metabolism, redox balance and Ca 2+ signaling during development, positioning mitochondria-bound hexokinase as a hub for intracellular signaling control. Abbreviations used: b-HB, b-hydroxybutyrate; b-HBM, b-hydroxybutyrate plus malate; 2-DG, 2-Deoxyglucose; Ap5A, P1,P5-Di(adenosine-5 0 ) pentaphosphate; DTNB, 5,5 0 -Dithiobis(2-nitrobenzoic acid); FCCP, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone; G6PDH, glucose-6phosphate dehydrogenase; mΔw, mitochondrial membrane potential; mETS, mitochondrial electron transport system; mPTP, mitochondrial permeability transition pore; mtHK, mitochondrial bound hexokinase; ODC, oxoglutarate dehydrogenase complex; Omy, oligomicyn; PDC, pyruvate dehydrogenase complex; ROS, reactive oxygen species; RRID, research resource identifier. 624