The blood-brain barrier (BBB) is a selective barrier. Other groups have demonstrated the ability of various cannabinoids to attenuate damage caused to the BBB in a range of pathologies. The role that AEA, and other endocannabinoids (eCBs), play in regulating BBB permeability has yet to be investigated using human cells, therefore, the aim of the present study is to establish whether they modulate BBB permeability. To model the BBB, co-cultures of human brain microvascular endothelial cells and human astrocytes were grown to confluence on Transwell collagen-coated inserts. BBB permeability was measured by transepithelial electrical resistance (TEER) using STX2 electrodes and an EVOM2 resistance meter. Resistance readings were taken at various intervals over 96h and compared to their baseline value. AEA (100 nM, 1 or 10µM) or other eCBs (10µM), and/or antagonist were added to the endothelial cells at 0 and again at 48h when the medium was replaced. In a separate series of experiments, inserts were subjected to 4h oxygen-glucose deprivation (OGD, to model ischaemia) by incubating them in GasPak EZ Anaerobe pouches with glucose-free RPMI medium. Reperfusion was established by returning the cells to normoxia and their specialised medium, and TEER was measured at various time-points over 28h. eCBs were added either pre- or post-OGD. Statistical analysis was conducted using one-way ANOVA with Dunnett’s post hoc test. Administration of AEA at 10µM only resulted in a significant, acute decrease in BBB permeability (i.e. an increase in barrier tightness) compared to baseline at 2, 50 and 52h (i.e. immediately after AEA administration; n=9; P<0.001-0.05). Receptor involvement was probed, and AM251 (CB1, 100nM), GW6471 (PPARα, 100nM) and GW9662 (PPARγ, 100nM) had not effect. However, the AEA-induced decrease in permeability was significantly inhibited by AM630 (CB2, 1μM) (P<0.001-0.05) and capsazepine (TRPV1, 1μM) (P<0.001-0.05). The decrease in permeability was also inhibited by URB597 (FAAH inhibitor, 1μM) (P<0.01-0.05) (n=6-8 for all antagonist studies). Of the other eCBs investigated, 2-AG, PEA and virodhamine did not alter permeability. However, NADA increased BBB permeability (P<0.001-0.01), whilst OEA decreased BBB permeability (P<0.05-0.001) (n=6-8). 4h OGD increased BBB permeability (P<0.001), but administration of AEA following OGD did not decrease permeability (n=7-12). However, AEA (100nM, 10 or 30µM) given pre-OGD promoted a more rapid decrease in permeability during reperfusion (P<0.001-0.05; n=5-7), as did OEA (P<0.001-0.05), PEA (P<0.05) and virodhamine (P<0.01-0.05) (n=5-6). 2-AG did not alter BBB permeability following OGD. In conclusion, this study demonstrates that eCBs do play a role in regulating BBB permeability in vitro using human cells.