Disruption of the blood-brain barrier (BBB) is a key factor in the development of stroke. N,N-dimethyltryptamine (DMT) is a hallucinogen alkaloid widely found in plants and animals. Beneficial effects of DMT was described in a rat stroke model, but BBB functions were not studied. Our aim was to examine the effect of DMT on the functions of the BBB following oxygen-glucose deprivation and reoxygenation (OGDR) in a rat primary BBB co-culture system.To model stroke in vitro, OGD was introduced for 6 hours on the BBB model, followed by reoxygenation for 24 hours with or without the addition of DMT. First, impedance measurement was performed to test the effect of OGDR on monocultures. To examine the barrier functions after OGDR a triple co-culture model on cell culture inserts was used. Transendothelial electrical resistance, sodium fluorescein and Evans blue-labeled albumin permeability was measured, and claudin-5 immunocytochemistry performed. After the OGDR treatment supernatants were collected to measure cytokine release.Impedance of rat brain endothelial cells (ECs), pericytes and glial cultures decreased in response to the OGD, but only ECs recovered to the control level at 24 h reoxygenation. Barrier functions were decreased and the proinflammatory cytokine release was increased after the OGDR. The DMT treated groups showed improved barrier functions and a decrease in cytokine release. These studies provide a background for the protective effect of DMT in stroke.The project was supported by: NKFIH OTKA-PD 137565 (forS.N.); ÚNKP-23-3 -SZTE-535 and the scholarship of the Gedeon Richter’s Talentum Foundation (for J.P.V.).