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The role of hydrogen peroxide and redox signaling was demonstrated in the regulation of peripheral vascular permeability, but it has been poorly studied for BBB. In our study, we demonstrated the effect of endogenous hydrogen peroxide on the permeability of the in vitro blood-brain barrier (BBB) model. Endogenous hydrogen peroxide was generated using a chemogenetic generator - D-amino acid oxidase (DAAO). The DAAO was delivered to endotheliocytes and astrocytes. It was demonstrated for the first time that the generation of hydrogen peroxide in individual cellular components of the BBB affects its permeability as a whole. The permeability of the model was determined by the diffusion rate of the fluorescent dye Lucifer yellow. It has been shown that the generation of endogenous hydrogen peroxide in both endotheliocytes and astrocytes increases the permeability of the model. After 24 hours of incubation with 5 mM D-alanine, in the case of peroxide generation in endotheliocytes, the permeability increased by 18%, and in the case of generation in astrocytes - by 36% compared to the initial value. After 48 hours of incubation with 5 mM D-alanine, the permeability was increased by 20% compared to the initial one both in the case of hydrogen peroxide generation in endotheliocytes and astrocytes.