Lymphocyte emigration from blood into peripheral lymph nodes is mediated by specialized high endothelial cells (HEC) lining the postcapillary venules. A current model for this process postulates that it occurs in three steps: weak, selectin-mediated interactions tether lymphocytes to the blood vessel wall; the lymphocytes are activated to increase the affinity of integrin-dependent adhesion and enhance motility; and finally the lymphocytes migrate actively across the endothelial cell layer. Some features of this model are simulated in vitro by cultured HEC, which support the adhesion and transmigration of lymphocytes. In particular, cultured HEC stimulate lymphocytes to change shape from spherical to polar. This shape change provides a convenient assay of the motility activation of lymphocytes. In this paper it is shown that this occurs without the lymphocytes becoming tightly adherent, but depends on contact with the endothelial cell surface. The shape change is labile: non-adherent polar lymphocytes removed from HEC revert to round with a half-time of less than 8 min. Reagents which block the interaction of L-selectin with its ligands inhibit the HEC-induced shape change; these include mannose-6-phosphate, fucoidan, polyphosphomannan ester, treatment of HEC with sialidases and an anti-L-selectin monoclonal antibody known to block its lectin function. The change in shape is partially inhibited by antisera to the L-selectin ligand GlyCAM-1. Thus it is concluded that in this in vitro system, L-selectin-mediated binding of lymphocytes to HEC is essential for optimal induction of the shape change. Lymphocytes change shape in response to cultured HEC without loss of surface L-selectin, although activation stimuli are known to promote shedding of neutrophil L-selectin as well as motility and increased adhesiveness. However, the lymphocyte change in shape is a reversible process, and this may have implications for the nature and sequence of the signals transmitted from endothelium to lymphocytes during homing to peripheral lymph nodes.