We have produced four mAbs reactive with the rat homolog of ICAM-1 (intercellular adhesion molecule-1), and presented evidence to indicate that the ICAM-1 molecule plays a differential role in the binding between high endothelial cells and lymphocytes, depending on the state of lymphocyte activation. The conclusion that the four antibodies (1A29, 6A22, 10A25, 10A56) specifically recognize the rat ICAM-1 homolog was based on: (i) their ability to inhibit homotypic aggregation of PHA-blasts; (ii) SDS-PAGE analysis of the antigen recognized; (iii) antigen distribution as assessed by immunoperoxidase staining of frozen sections; and (iv) cytokine-induced up-regulation of the antigen. Involvement of the ICAM-1 molecule in lymphocyte binding to high endothelial cells, the vital step in lymphocyte extravasation in lymph nodes, was examined by the use of one of the newly developed antibodies, and it was found that binding of activated but not resting lymphocytes to cultured high endothelial cells was significantly blocked by the anti-ICAM-1, implying that ICAM-1 has differential involvement in the adherence of lymphocytes to high endothelial cells and that it may play an important role in dissemination of memory lymphocytes to systemic circulation by facilitating or strengthening the binding to the specialized postcapillary high endothelial venules (HEV).