CD44 is a transmembrane glycoprotein found on a variety of cells including those of myeloid and lymphoid origin. CD44 is highly conserved among various species and is involved in the homing of lymphocytes and monocytes to lymph nodes, Peyer's patches, and sites of inflammation. In the present study, we demonstrate that monoclonal antibody (mAb) 9F3, directed against murine CD44 expressed on cytotoxic T lymphocytes (CTls), can trigger the lytic activity of CTls and redirect CTl-mediated lysis to antigen-negative Fc receptor-positive target cells. Similar redirected lysis was also inducible using mAb MEL - 14, directed against the lymphocyte homing receptor for endothelium (gp - 90MEL-14). The redirected lysis induced by mAbs 9F3 and MEl-14 in the CTL was similar to that induced by mAbs against the aβ T-cell receptor or CD3. In contrast, mAbs directed against CDS, CD45R, and CD11a (LFA-1, lymphocyte function-associated antigen 1) failed to evoke lytic activity. Furthermore, CD44 and MEl-14 mAbs were able to mediate NK cell lysis of the NK-resistant tumor PS15. The current study demonstrates that CD44 and gp_90MEL-14 molecules, in addition to participating in T-cell homing and adhesion, may play a major role in delivering the transmembrane signal to the CTl that triggers the lytic activity, even when the T cell receptor is not occupied. The alternate pathway of CTL activation characterized in this study may exhibit both beneficial and deleterious effects on the host. On one hand, this property may enable CTL to kill cancer cells or virally-infected cells which may fail to express major histocompatibility complex (MHC)-encoded antigens. On the other hand, this alternate pathway may contribute to nonspecific tissue damage seen at sites of inflammation.