High endothelial venules (HEV) are specialized vessels that support abundant lymphocyte emigration from peripheral blood into secondary lymphoid organs. HEV endothelial cells (HEVEC) exhibit particular structural and functional features, including secretion of the HEV-specific extracellular matrix protein hevin and an array of uniquely glycosylated counter-receptors for L-selectin expressed on lymphocytes. These ligands are collectively called the peripheral lymph node addressin (PNAd), originally defined by the monoclonal antibody MECA-79. PNAd expression was used to purify HEVEC by positive immunoselection from enzyme-digested human tonsils after negative immunoselection for other cells. Purified HEVEC maintained secretion of hevin and homogenous expression of intercellular adhesion molecule (ICAM)-1 (CD54), ICAM-2 (CD102), and CD31, at high levels following 8 days in culture. Expression of functional PNAd was maintained during the first 4 to 5 days of culture but decreased gradually and disappeared on day 8, while the expression of CD34 remained strong. However, the CD34 glycoform shifted toward the in situ phenotype of flat-walled vessels, suggesting that the observed loss of L-selectin binding determinants and MECA-79 antigen was due to down-regulation of the glycosyl- and sulfo-transferases essential for their expression. Our rapid and reproducible method to establish HEVEC cultures provides a useful mechanistic tool for identification of the factors that induce and maintain the HEV phenotype, as well as a source for isolation of HEV-specific genes.