We have developed an improved intralumenal digestion method to get a long-term primary culture of rat lymphatic endothelial cells (rLECs) that maintained their original phenotypes. rLECs in vitro under hypoxia retained their original lymphatic properties observed in the thoracic duct. Blocking Notch signal with a gamma-secretase inhibitor and transfection of a cDNA expressing a dominant negative form of Delta1 indicated that Notch signal downregulated VEGFR-2 under hypoxia and inhibited cell migration. These findings indicated that Notch signal was still operative in mature lymphatic endothelial cells in response to the oxygen concentration.