Naive T cells mount a vigorous proliferative response to superantigen (SAg) stimulation in vivo. The proliferative response is followed by a partial deletion of responder T cells. Part of the deletion process has recently been attributed to the action of regulatory cytotoxic T cells that recognize major histocompatibility complex (MHC) class I-associated antigen receptor determinants on the target cell surface. Responder T cells that survived the SAg response were found to be incapable of generating a secondary proliferative response to a SAg challenge. We show here that this 'anergy' is enforced by CD8-positive regulatory suppressive T cells. These regulatory cells inhibit cell division of preactivated T cells but not the Sag response of naive T cells. Regulatory T cells are not generated in the presence of cyclosporin A and, once activated, become inactivated or deleted when restimulated in the presence of this immunosuppressive drug.