The constitutive recirculation of lymphocytes between the widely distributed organs of the immune system is essential for host defence. We have developed an in vitro model of lymphocyte migration from the blood into lymph nodes which employs primary cultures of high endothelial cells (HEC). HEC-adherent lymphocytes adopt one of two distinct morphologies which correlates with their position in the endothelial layer; type I cells are bound to the surface of HEC and type II cells are underneath the endothelial layer. In a previous study we reported that the numbers of type I and type II cells are independently regulated, however the relationship between these two lymphocyte populations was not determined. In this study we have carried out detailed kinetic, phenotypic and functional analyses of type I and type II lymphocytes and determined their relationship. Using allotype marked lymphocytes from the PVG.RT7a and PVG.RT7b rat strains in a pulse-chase analysis, type I and type II lymphocytes were found to represent the same population of lymphocytes at different stages of interaction with the endothelial layer, rather than representing two independent lymphocyte populations. Migration was an irreversible event and the efficiency of migration (i.e. transition from type I to type II) was related to the concentration of lymphocytes plated on to the HEC layer. Following transmigration lymphocytes showed an increased ability to migrate across HEC layers and to bind to immobilised CS1 peptide. The increased binding to CS1 peptide was transient and fell to control levels over a 3 hour time period. The expression of alpha 4 integrin subunit on lymphocytes was unchanged following migration which suggests that the affinity of the CS1 receptor, alpha 4 beta 1, is upregulated by interaction with HEC. Together these results suggest that transendothelial migration is regulated by increases in the affinity of alpha 4 beta 1 integrin on lymphocytes following contact with HEC.