A family of caspases has been implicated as an effector in various forms of apoptosis. The present study investigated whether this family of proteases is involved in the induction of intrathymic clonal deletion in comparison with apoptosis induced in the thymus by various signals. Potent apoptosis of thymocytes was induced in fetal thymus organ cultures (FTOC) when FTOC were treated with glucocorticoid, radiation, and anti-CD3 monoclonal antibody (mAb). As a model of negative selection based on apoptotic clonal deletion, the elimination of Vbeta8-expressing thymocytes was induced by inoculating Staphylococcal enterotoxin B (SEB) into FTOC. Addition of a peptide-based caspase inhibitor resulted in the protection of thymocytes from apoptosis induced by glucocorticoid, radiation, and anti-CD3 mAb. In contrast, the same treatment failed to prevent clonal deletion of Vbeta8high thymocytes. These results suggest that different pathways of cell death operate in the thymus that may be distinguished depending on the caspase/protease utilized in each pathway.