The present study was undertaken to determine if neuropeptide Y (NPY) induces proliferation of rat aortic endothelial cells (RAECs). Since NPY increased the permeability of RAEC monolayers to large molecules via the NPY Y₃-receptor, RAEC proliferation has been evaluated in terms of NPY receptor subtypes, and also intracellular mechanisms. RAECs were incubated with gases containing 20, 15, or 10 % O₂, and a certain amount of N₂ depending on the O₂ content in 5 % CO₂-incubators. NPY (10^-9 -10^-6 M) increased the RAEC numbers under hypoxic conditions, such as 15 or 10 % O₂. Peptide YY elicited no proliferative effect on RAEC, and NPY_(18-36) inhibited the NPY-induced increase in cell number, suggesting that NPY increases the RAEC count through the NPY Y₃-receptor. Pertussis toxin, U-73122, GF-109203X, myristorylated AIP, and Wortmannin inhibited the NPY-induced proliferation of RAECs concentration-dependently. DY9760e little affected the proliferation caused by NPY. ML-9 and imatinib actually enhanced the NPY-induced proliferation of cells. These results indicated that the NPY Y₃-receptor is coupled with G_i-protein, and that NPY-induced increases in RAEC proliferation are mediated by phospholipase C - protein kinase C and/or phosphatidyl-inositol-3 kinase pathways. In intracellular Ca⁺⁺-calmodulin dependent pathways, calmodulin-dependent protein kinase II partly participates in the NPY-induced cell proliferation. Regarding the previously-reported effect of NPY on the permeability of RAEC monolayers to large molecules, it is probable that protein kinase C and phosphatidyl-inositol-3 kinase pathways are activated for both permeability and cell proliferation induced by NPY under hypoxia, relevant to new insights into the roles of NPY in ischemia/hypoxia.