The CD95 (APO-1/Fas) ligand (CD95L) mediates apoptosis in sensitive target cells, Ca(2+)-independent cytotoxicity of cells from perforin knock-out mice, and peripheral deletion of activated T cells through engagement of its cognate receptor CD95. Double-positive thymocytes show a high constitutive expression of CD95. Therefore, we used a model system and investigated whether negative selection through apoptosis might involve CD95/CD95L. We analyzed whether CD95L may induce antigen-specific deletion of double-positive thymocytes from mice transgenic for a lymphocytic choriomeningitis virus (LCMV)/H2b-specific T cell receptor (TCR). These cells are deleted in vitro upon addition of the LCMV-peptide 33-41 in a major histocompatibility complex-class I-restricted fashion. Deletion was not blocked by soluble mouse and human CD95-Fc receptor decoys. CD95-Fc receptor decoys, however, were effective in blocking apoptosis induced by mouse CD95L-transfected L929 cells in sensitive CD95+ target cells and in thymocytes. These results suggest that TCR-induced deletion of immature thymocytes in vitro is independent of CD95L. Thus, our data argue against a role of CD95L in negative selection of MHC-class I-restricted autoreactive thymocytes.