The specific pattern of lymphocyte localization and recirculation is important for the induction and expression of normal immune responses. In order to home to lymph nodes (LN), lymphocytes must first recognize and bind to specific high endothelial venules (HEV) in the LN. Binding to LN HEV is mediated by specific lymphocyte receptors, termed homing receptors, which are recognized by the mAb MEL-14. We examined the changes that occur in homing receptor expression after activation of murine T lymphocytes in vitro. Cells activated in MLC or by Con A undergo a 75% loss in their ability to recognize HEV, as demonstrated by a decrease in binding to HEV in vitro. Large, activated cells isolated from a primary MLC by elutriator centrifugation were completely unable to recognize HEV, whereas the small cells in the same culture continued to bind well. Flow cytometric analysis with MEL-14 showed that the activated fraction had lost expression of gp90MEL-14, the homing receptor Ag, whereas the inactivated cells remained MEL-14+. Concomitant with the loss of homing receptor expression, most of the activated cells became strongly peanut agglutinin (PNA)-positive, demonstrating a marked change in surface glycosylation. Thus, these MLC consist of two major populations of T cells--small, inactivated lymphocytes that are MEL-14+PNAlo and large, activated blast cells that are MEL-14-PNAhi. Purified MEL-14+ T cells activated by Con A gave rise to MEL-14- progeny, showing that gp90MEL-14 is lost from gp90MEL-14-positive precursors, rather than from the selective growth of MEL-14- cells. Furthermore, the loss of Ag expression on at least some activated cells is reversible in resting culture, with almost half of the cells reverting to MEL-14+ after the cessation of stimulation. These experiments show that activation of T cells results in down-regulation of surface homing receptors, resulting in their inability to recognize and bind to the endothelial surface of HEV. This suggests that the activation of T cells in vivo would result in a dramatic and physiologically significant change in their migration and localization properties which would be important during a normal immune response.