The role of B7 co-stimulatory signaling in in vivo tumor rejection remains incompletely characterized. In particular, the relative competence of B7-1 (CD80) and B7-2 (CD86) to provide effective co-stimulus is not well defined, and the identification of the T cell co-stimulatory receptor that mediates B7 co-stimulation in tumor rejection has not been addressed. These issues were studied by assessing rejection of B7-negative or B7-transfected tumor cells in CD28-expressing or CD28-deficient hosts. B7-negative EL4 tumor cells grew progressively in normal syngeneic C57BUL6 (B6) mice. In contrast EL4 cells transfected with either full length B7-1 or full length B7-2 were rejected, indicating that both B7-1 and B7-2 are competent to mediate rejection of EL4 tumor cells. Expression of truncated B7-1 or B7-2 products, with complete deletion of cytoplasmic domains, was as effective as expression of full length B7-1 or B7-2 in mediating rejection. In contrast to the rejection of B7-transfected EL4 cells observed in CD28-expressing syngeneic hosts, B7-1- and B7-2-positive EL4 cells as well as control EL4 cells grew progressively in CD28-deficient mice, demonstrating the requirement for host expression of CD28 in B7-mediated tumor rejection. These results indicate that interaction of host CD28 with co-stimulatory extracellular B7-1 or B7-2 ligands expressed on tumor cells can play a necessary role in mediating tumor rejection.