The lpr gene induces in mice, accumulation of large numbers of CD4-CD8- (double negative [DN]) T lymphocytes which bear adhesion molecules not characteristic of normal resting T cells. These cells fail to acquire interleukin 2 (IL-2) receptors, produce IL-2, and proliferate when activated with mitogens or monoclonal antibodies (mAbs) against the T cell receptor (TCR). Because of these poor functions in vitro, the nature and significance of DN T cells in the autoimmune disease process is not clear. In the current study, we describe a surprising finding that mAbs against CD3-TCR-alpha/beta complex can strongly trigger the lytic activity of the DN T cells to induce redirected lysis of Fc receptor-positive targets. Similar redirected lysis was also inducible using mAbs against CD44 and gp90MEL-14, molecules involved in the binding of lymphocytes to endothelial cells. The spontaneous cytotoxic potential of the DN T cells was further corroborated by demonstrating that the lpr DN T cells constitutively transcribed perforin gene but failed to express granzyme A. The current study suggests that DN T cells are capable of mediating lysis of autologous cells bearing the specific ligands for adhesion molecules involved in the signaling of cytotoxicity. These findings provide a novel insight into the functional significance of DN T cells in lpr mice and their potential role in the pathogenesis of autoimmune disease.