Large-scale extravasation of lymphocytes takes place in vivo under physiological conditions in lymph nodes at very specialized vascular segments called high endothelial venules (HEV). When circulating lymphocytes leave the blood, they first bind to endothelial cells of HEV (HE cells) and subsequently enter lymph nodes by crossing the endothelial lining of HEV. Although the lymphocyte-HEV interaction has recently been the subject of intense research by many laboratories, it has been studied almost exclusively by the use of the lymphocyte-binding assay in which lymphocyte binding is examined on nonviable HEV present on frozen sections and, hence, no dynamic interaction between HE cells and lymphocytes has been studied. We report herein that endothelial cells of rat HEV can be grown in vitro and that the lymphocyte-HEV interaction can be studied dynamically using viable cells in culture vessels. The identification of the cultured line, termed Ax, as HE cells was based on their phenotypic, morphological, cytochemical and biochemical characteristics, and most importantly on its in vitro behavior, particularly in terms of its specific ability to interact with mature lymphocytes. Phenotypic analysis demonstrated that not only did monoclonal antibodies, known to react with HE cells, recognize the Ax but also a monoclonal antibody raised against the Ax specifically recognized HE cells in vivo, as determined by an immunoperoxidase staining of frozen sections, supporting the notion that the cell strain, Ax, is derived from HEV. This Ax, even after long-term culture (greater than 50 passages), allowed mature, but not immature, lymphocytes to bind to the cell surface and subsequently transport bound cells underneath their cytoplasm. This phenomenon was inhibited in a dose-dependent manner by various reagents known to inhibit lymphocyte recirculation in vivo. The cultured line derived from HE cells should provide a means to investigate the biochemical nature of lymphocyte-HEV interaction, and to understand the molecular mechanisms underlying the large-scale lymphocyte traffic taking place in vivo.