Activation of antigen specific T cells requires more than stimulation through the TCR-CD3 complex. A second or costimulatory signal is also required, and this second signal can be delivered by interactions between CD28 and B7, ligands expressed on T cells and antigen presenting cells respectively. We have examined the role of the CD28-B7 interaction in superantigen mediated T cell activation and intrathymic negative selection by blocking B7 molecules with a high affinity soluble ligand, CTLA4lg. In vitro T cell activation mediated by both virally encoded endogenous and exogenous bacterial superantigens was significantly blocked by the addition of CTL4Alg to cultures. However, intrathymic clonal deletion in vivo and in fetal thymic organ cultures was not inhibited by blocking B7 molecules. Therefore, although the CD28-B7 costimulation pathway is necessary for T cell activation, it does not appear to play a role in intrathymic clonal deletion.