The costimulatory ligands B7-1 and B7-2 are expressed on the surface of antigen-presenting cells and interact with the costimulatory receptors CD28 and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) expressed on T cells. Although B7-1 and B7-2 are homologous ligands having common receptors, they exhibit distinct biochemical features and roles in immune regulation. Several biochemical and structural studies have indicated differences in the oligomeric state of B7-1 and B7-2. However, the organization of B7 ligands on the cell surface has not been examined. By using photobleaching-based FRET (pbFRET), we demonstrate that B7-1 and B7-2 adopt different oligomeric states on the cell surface. Our study shows that B7-2 exists as a monomer on the cell surface whereas B7-1 exists predominantly as dimers on the cell surface. A series of mutations in B7-1 result in the expression of a predominantly monomeric species on the cell surface and validate the dimer interface proposed by prior crystallographic analysis. The difference in the oligomeric states of B7-1 and B7-2 provides insight into the geometric organization of the costimulatory receptor-ligand complexes in the immunological synapse and suggests constraints on signal transduction mechanisms involved in T cell activation.