Mice homozygous for lpr and gld develop lymphadenopathy characterized by the progressive accumulation of an unusual population of CD4-, CD8-, CD2-, IL-2R- double-negative (DN) T cells that express reduced levels of TCR-alpha/beta, high levels of CD45 (B220) and Ly-6C and variable levels of CD69. These cells are refractory to most stimuli, including staphylococcal entertoxins and cross-linking of the TCR, Ly-6C, and CD69. For normal T cells, the binding of ligand to the TCR alone is insufficient to induce a proliferative response and can result in the induction of a state of prolonged anergy. Efficient stimulation is dependent on the delivery of a second or costimulatory signal. Recently it was reported that CD28 can provide costimulatory signals to T cells and, that these signals can prevent anergy induction in T cell clones. We investigated the possibility that lpr and gld DN T cells are unresponsive because they fail to transduce signals via CD28. These studies showed that highly purified B220+ TCR-alpha/beta+ DN T cells expressed high levels of CD28, responded weakly to stimulation with PMA and anti-CD28 mAb and quite strongly to PMA, anti-CD28 antibody and high concentrations of immobilized anti-TCR-alpha/beta antibodies. The latter stimulus also induced low levels of expression of CD2 and IL-2R and secretion of modest amounts of IL-2. Although DN T cells proliferated and secreted IL-2, these responses differed qualitatively and quantitatively from those of +/+ and lprB220- T cells. Consistent with its effects on normal T cells, cyclosporin A partially inhibited the response of DN T cells to TCR cross-linking and CD28 ligation. Studies of synergism between CD28-, Ly-6C-, and CD69-mediated signals revealed that ligation of CD28 enhanced the proliferative response induced by cross-linking of Ly-6C or CD69 on +/+, lpr and gld B220- T cells but had no effect on the unresponsiveness of DN T cells to these stimuli. Ligation of CD28 did not reverse the unresponsiveness of DN T cells to SEB and had only a weak synergistic effect on the response of B220- T cells. Together, these observations suggest that the mechanisms leading to immunosuppression of DN T cells are complex and appear to involve abnormalities in signal transduction via the TCR and CD28 and possibly via Ly-6C and CD69 as well.