Costimulation of T cell proliferation can occur through the CD28 signal transduction pathway. In addition, other cell surface receptors, including the CD28 homolog CTLA-4, have been proposed to be capable of providing costimulatory signals. We have examined the response of CD28-deficient T cells to activation by a variety of agonists. We demonstrate that proliferation of CD28-deficient T cells in the presence of antigen-presenting cells or B7-1 transfectants is markedly reduced. Although CTLA-4 can be expressed on CD28-deficient T cells, we observed no B7-dependent costimulation in the absence of CD28. This data demonstrates that CD28 is the major B7-binding costimulatory ligand on T cells. Furthermore, our data suggest that CD28 is the primary, and perhaps exclusive, costimulatory receptor used by traditional antigen-presenting cells to augment the proliferation of antigenactivated T cells.