Programmed cell death (apoptosis) is crucial for thymocyte development. We analyzed the role of the ubiquitin (Ub)-proteasome pathway in dexamethasone-triggered and TCR-mediated apoptosis in fetal thymic organ culture (FTOC). Proteasome activity was increased in apoptotic thymocytes, as visualized by active-site labeling of proteasomal beta subunits. The activity of deubiquitinating enzymes in murine apoptotic thymocytes was likewise examined by active-site labeling. We show that the deubiquitinating enzyme USP7 (HAUSP) is proteolytically processed upon dexamethasone-, gamma-irradiation-, and antigen-induced cell death. Such processing of HAUSP does not occur in caspase 3-/- thymocytes, or upon pretreatment of wild type thymocytes with the general caspase inhibitor ZVAD-fmk. Thus, our results suggest that thymocyte apoptosis leads to modification of deubiquitinating enzymes by caspase activity and may provide an additional link between the ubiquitin-proteasome pathway and the caspase cascade during programmed cell death.