Making use of mice deficient for terminal deoxynucleotidyl transferase (TdT) expression and a random peptide library, we have examined the diversity and peptide specificity of the neonatal T cell repertoire specific for a single H-2Db-restricted peptide. Consistent with the predicted decrease in repertoire diversity, polyclonal CTL lines and individual clones from different TdTo mice are more similar to each other than those from different wild-type mice in terms of their fingerprints of cross-reactivity to the library and their TCR sequences. We have also found that several TdTo CTL clones cross-react with many more library peptides than wild-type CTL clones. In a few instances, the degree of peptide promiscuity correlates with TCR sequence characteristics such as N region addition and homology-directed recombination, but not CDR3 loop length. Based on epitope titrations for each clone, TCR affinity for antigen is consistently high; thus, this reduced specificity for peptide may coincide with an accentuated affinity for the alpha helices of the MHC. Peptide promiscuity in the neonate may allow the relatively small numbers of T cells in the periphery to protect against a broader range of pathogens.