The B cell activation antigen B7/BB1 has been shown to co-stimulate growth of human T cells by binding the T cell molecule CD28. In mice, the heat-stable antigen (HSA) has also been shown to act as a co-stimulator for T cell growth. In this study, we have evaluated the contributions of B7 and HSA to the co-stimulatory activity of antigen-presenting cells (APC). Mouse B7 provides co-stimulatory activity for murine CD4 T cells in anti-CD3-induced proliferation. Human CTLA4Ig, a chimeric molecule comprising the extracellular region of CTLA-4 fused to an immunoglobulin C gamma fragment, binds to murine B7. We, therefore, use human CTLA4Ig and the hamster anti-HSA monoclonal antibody 20C9 to analyze the relative contributions of B7 and HSA to the co-stimulatory activity of murine spleen APC. Our data reveal that both murine B7 and HSA are expressed by dendritic cells and by low-density spleen B cells. Either CTLA4Ig alone or anti-HSA alone inhibited CD4 T cell proliferation to anti-CD3 by > 90%, while CTLA4Ig and anti-HSA together were far more efficient in inhibiting clonal expansion of CD4 T cells. These results demonstrate that functionally defined co-stimulation involves at least B7 and HSA and suggest that signals delivered by B7 and HSA synergize in promoting T cell growth.