Extravasation of leukocytes from the blood is essential for normal lymphocyte recirculation as well as in mounting an adequate inflammatory response in different tissues. Leukocyte migration from the blood is controlled by sophisticated interactions between surface receptors on leukocytes and their corresponding endothelial cell ligands. Here we describe a novel adhesion molecule, lymphocyte-vascular adhesion protein-2 (L-VAP-2), recognized by 4G4 mAb that was produced by immunizing mice with an enriched endothelial cell preparation isolated from inflamed human synovium. mAb 4G4 stains a subpopulation of venules in lymphoid and nonlymphoid tissues as well as a few high endothelial venules in lymphoid tissues. L-VAP-2 is constitutively expressed on human umbilical vein endothelial cells, and its expression cannot be up-regulated by cytokines and mitogens such as TNF-alpha, IL-1, and LPS. The Ag is expressed on approximately 20% of PBL, whereas granulocytes and monocytes are negative. On lymphocytes, L-VAP-2 is preferentially expressed on B cells and CD8+ T cells. The molecular mass of L-VAP-2 is approximately 70 kDa as determined from immunoprecipitated, 125I-labeled endothelial cell lysates. The involvement of L-VAP-2 in lymphocyte binding to endothelium was tested in vitro using human umbilical vein endothelial cells. Both the intact antibody and F(ab')2 fragments of it consistently inhibited lymphocyte binding to human umbilical vein endothelial cells by approximately 25%. On the basis of the molecular mass estimation and the staining of tissue sections, leukocyte populations, and ICAM-1 and ICAM-2 transfectants, L-VAP-2 appears to be a novel Ag involved in the lymphocyte-endothelial cell interaction.