How negative selection shapes a polyclonal population of self-reactive T cells has been difficult to address directly because of the lack of means to isolate T cells reactive to a particular self-peptide. Here, using mice transgenic for the TCR-β chain of a CTL clone directed against a male-specific peptide, we compared the preimmune repertoire reactive to this peptide in male and female animals. Surprisingly, in the presence of the deleting ligand, as many as 25%–40% of reactive T cells escaped clonal deletion. A correlation was found between T cell avidity, TCRα structures, and susceptibility to negative selection. These results suggest that numerous low-affinity self-specific T cells persist in the periphery and show that a deleting ligand can specifically narrow the structural diversity of the TCR repertoire.