Previously in-vivo studies showed that neuropeptide Y (NPY) elevates vascular permeability in isolated lung perfusion preparations, possibly through binding to the NPY Y₃ receptor. The present study was undertaken to test the hypothesis that NPY directly impacts on rat aortic endothelial cells (RAECs), using monolayers in double-chamber culture method under conditions of normoxia (5 % CO₂ + 20 % O₂ + 75 % N₂) or hypoxia (5 % CO₂ + 5 % O₂ + 90 % N₂). RAECs were cultured on the base of the upper chamber, into which FITC-labeled albumin was introduced, and permeation into the lower chamberwas measured. The RAEC monolayer was treated with NPY, in a concentration range from 10^-8 M to 3 x 10^-7 M, for 2 h under conditions of normoxia or hypoxia. With hypoxia, NPY concentration-dependently increased the permeability of the RAEC monolayer, whereas with normoxia no significant change was observed. Peptide YY, NPY Y₁ and NPY Y₂ agonists, and NPY Y₁ antagonist exerted no significant effects under hypoxic conditions. NPY_18-36, an NPY Y₃ antagonist, elicited an inhibitory action on NPY-induced increase in monolayer permeability. Furthermore, neither L-NMMA, a nitric oxide (NO) synthetase inhibitor, bradykinin B₂-receptor antagonist FK3657, nor vascular endothelial growth factor (VEGF) receptor-coupled tyrosine kinase inhibitor Tyrphostin SU-1498, injected into the medium of upper chamber, affected the NPY-induced permeability changes under hypoxic conditions. The results suggested that NPY increases the permeability across the RAEC monolayer in a close relation with low oxygen tension, possibly mediated by direct action on the NPY Y3-receptor expressed on the endothelial cell membrane. Furthermore, this NPY-induced increase is not likely due to either NO, bradykinin or VEGF.