Systemic administration of high doses of soluble Ag induces peripheral CD4+ T cell tolerance in unmanipulated hosts. To test whether tolerance is modified under conditions of transient lymphopenia, we tracked the response of 5C.C7 TCR-transgenic CD4+ T cells to i.v. moth cytochrome c peptide in mice that received low-dose gamma irradiation 10 days previously. This model was chosen because it does not support spontaneous lymphopenia-induced proliferation of 5C.C7 cells, allowing the study of Ag-specific responses without interference from simultaneous spontaneous proliferation. Clonal expansion in response to i.v. peptide was increased in irradiated mice, while clonal deletion was severely impaired in comparison with untreated animals. Amplified TCR triggering was observed in irradiated hosts, consistent with dendritic cell activation leading to enhanced Ag presentation. Failure of deletion was accompanied by persistent T cell activation and accumulation of Th1 effector cells. Up-regulated expression of IL-7R and the prosurvival protein Bcl-x(L) was associated with clonal persistence. Cells with memory and naive phenotypes were both represented within persistent clones, but no Th1 function could be demonstrated within the long-term memory population. Failure of clonal deletion in irradiated hosts represents a novel mechanism limiting TCR diversity in a lymphopenic environment and may contribute to subsequent autoimmunity.