AbstractIn this study we report the characterization of monoclonal antibody (mAb) 8B4/20, raised against immature human thymocytes, that identifies a novel leukocyte antigen. The molecular characterization of the antigen by immunoprecipitation and immunoblotting yields, under nonreducing conditions, a specific band of 120 kDa which, under reducing conditions, displays a slightly lower molecular mass (110 kDa). mAb 8B4/20 detects a molecule found on the majority of thymocytes with an inverted gradient of expression when compared to CD3. It appears at high density on the CD3−/lowthymocytes, at reduced density on the CD3medand double‐positive thymocytes, and is absent on CD3hiand singlepositive thymocytes and on peripheral blood T cells. Immunohistochemistry on frozen sections demonstrates cortical staining of the thymic lobules. Flow cytometric analysis of the different subsets of peripheral blood mononuclear cells shows that mAb 8B4/20 detects an antigen expressed only on CD56+/CD16+natural killer cells and on a fraction of CD14+monocytes. T cells, B cells, erythrocytes, granulocytes and platelets are consistently negative. The expression of the molecule on tumor cell lines does not show lineage restriction. Analysis of phytohemagglutinin plus recombinant interleukin‐2‐activated peripheral blood lymphocytes shows that mAb 8B4/20 identifies an antigen expressed on CD3+cells by week 3 of culture. Thus, it recognizes a very late activation antigen (VLA) on mature T cells. The cell distribution and the electrophoretic pattern of the molecule identified by mAb 8B4/20 is distinct from that of known CD and of integrin/VLA molecules. Its function on thymocytes is so far unknown; however, the binding of mAb 8B4/20 to tumor lines induces changes in the morphology and adhesive properties of the 8B4/20+cells growing in suspension. We suggest that mAb 8B4/20 recognizes a molecule that may be involved in interactions between thymocytes and other thymic structures that may be relevant for the selection