Negative selection in thymus occurs by apoptosis in CD4+CD8+ cells. These immature thymocytes are readily killed, both in vitro and in vivo, by glucocorticoid treatment. Increased levels of intracellular cAMP in vitro also induce apoptosis of thymocytes and T cell receptor (TcR) stimulation potentiate cAMP responses through receptors linked to adenylic cyclase. Presently, we have tested the possibility that TcR-mediated apoptosis in vivo may require the glucocorticoid receptor (GR) as a downstream, intracellular mediator. Use of the GR antagonist RU-486, 24 h before and simultaneous with, anti-CD3 or 5'-(N-ethyl)-carboxamide-adenosine (NECA) treatment, resulted in a selective inhibition of CD4+CD8+ thymocyte death. In addition, a low dose of glucocorticoid potentiated thymocyte death induced by anti-CD3 monoclonal antibodies. These data support a model in which thymic negative selection depends on a defined set of transduction signals which potentiate the GR to become responsive to endogenous levels of glucocorticoid.