We have found suppressor T cells that inhibit the proliferative response of naive CD4(+) T cells in T cell receptor (TCR) Vbeta8.1 transgenic mice rendered tolerant in vivo by inoculation of Mls-1(a)-positive cells. This suppression was mediated by CD4(+) T cells but not by CD8(+) T cells or double-negative (DN) cells, and splenic CD4(+) T cells from tolerant mice displayed a greater suppression than lymph node CD4(+) T cells. Cell contact was required for efficient suppression, and known inhibitory cytokines such as IL-4, IL-10, and transforming growth factor beta were not involved. Suppressor T cells inhibited IL-2 production by naive CD4(+) T cells, and the addition of exogenous IL-2 diminished the suppressed activity while having little activity on tolerant T cells. Suppression was abolished by the elimination of CD25(+) T cells in the tolerant CD4(+) T cell subset. CD25(+)CD4(+) T cells suppressed the proliferative response of the residual fraction of the nonanergic population, namely, 6C10(+)CD4(+) T cells still present in the tolerant mice. However, 6C10(-)CD4(+) T cells still had reduced reactivity to Mls-1(a) even after CD25(+)CD4(+) T cells were removed and exogenous IL-2 was added. Suppressor cells appear to affect only residual nonanergic cells in situ, thereby facilitating the maintenance of the unresponsive state in vivo. These data provide a framework for understanding suppressor T cells and explain the difficulties and variables in defining their activity in other systems, because suppressor T cells apparently control only a small population of nonanergic cells in the periphery and may be viewed as a homeostatic mechanism.