Circulating lymphocytes leave the blood by binding to specialized high endothelial cells lining postcapillary venules in lymphoid organs or sites of chronic inflammations, migrating through the vessel wall into the surrounding tissue. The capacity of lymphocytes to recognize and bind to high endothelial venules (HEVs) is thus central to the overall process of lymphocyte traffic and recirculation. We show that viable human lymphocytes bind selectively to HEVs in frozen sections of normal human lymph nodes, thus defining a simple in vitro model for the study of human lymphocyte homing properties. Optimal conditions for the quantitative analysis of lymphocyte-HEV interaction are described. Furthermore, by using this assay, we demonstrate that the ability of human lymphocyte populations to bind to HEVs parallels their presumed migratory status in vivo. Thus, thymocytes and bone marrow cells, which are sessile in vivo, bind poorly to HEVs in comparison with mature circulating lymphocytes in peripheral blood or in peripheral lymphoid tissues. These results indicate that HEV-binding ability is a regulated property of mature lymphocytes and, as demonstrated previously in animal models, probably plays a fundamental role in controlling lymphocyte traffic in humans. The in vitro model of lymphocyte-HEV interaction thus provides a unique means to assay the migratory properties of normal and neoplastic human lymphocyte subsets, to analyze the role of lymphocyte traffic mechanisms in normal and pathologic inflammatory reactions, and to define some of the molecular mechanisms responsible for the control of lymphocyte migration and positioning in humans.