To define T cell co-stimulatory molecules that work in the early phase of T cell activation, we established monoclonal antibodies (mAb) that inhibit or enhance T cell activation by the histiocytic leukemia cell line U937. One of the mAb, 53H5, which recognized both T cells and U937, was identified to bind to CD82 by expression cloning. Functional analyses of CD82 revealed that 1) CD82 needs to exist on both T cells and U937 for the full activation of T cells; 2) CD82 expression is up-regulated on both T cells and U937 by stimulation such as CD3 ligation or treatment with phorbol 12-myristate 13-acetate; 3) overexpression of CD82 enhances both homotypic and heterotypic cell adhesion between T cells and U937; 4) CD82 signal co-stimulates T cells and the signal works synergistically with the CD28-mediated T cell co-stimulation signal; 5) in mixed leukocyte reactions using U937 as stimulator cells, CD82 overexpression on U937 correlates with the higher allogeneicity of U937 cells. These results indicate that CD82 co-stimulates T cells not only by sending intra-T cell signals that work synergistically with CD28 signals but also by inducing enhanced T cell-antigen-presenting cell interaction.