T cell receptors (TCRs) and immunoglobulins (Igs) derive a large fraction of their repertoire from diversity generated at the junctions of the V, D, and J coding segments. This diversity is derived both from the random deletion of nucleotides from the ends of coding regions and from the subsequent addition of nontemplated N region nucleotides. While the vast majority of TCRs and Igs from adult mice have N regions, less than 5% of both TCR-gamma/delta and Ig from fetal and neonatal mice have N regions. This study analyzed the ontogeny of junctional diversity of TCR-alpha/beta. Genomic DNA or C beta-primed cDNA was prepared from thymocytes of mice at varying stages in ontogeny, and the rearranged V beta 8 or V beta 5 sequences were amplified by polymerase chain reactions. Sequencing of the V beta-D beta-J beta junctions showed few N regions early in ontogeny, although the fraction of sequences with N regions exceeded that previously reported for Ig and for TCR-gamma/delta. N regions were found in 13% of V beta junctional sequences from day 18-19 fetal thymocytes, 33% of sequences from newborn thymocytes, 76% of sequences from day 4 postnatal thymocytes, and 88% of sequences from 5-wk-old thymocytes. In addition, nonrandom usage of the D beta and J beta segments was observed in both fetal and adult TCR sequences. While the usage of each of the six J beta 2 segments was different, the same pattern of usage was seen regardless of whether D beta 1 or D beta 2 was used, suggesting that a factor controlling the rate of usage of each J segment is intrinsic to the J gene itself. Since TCRs derive so much of their diversity from N regions, the relative paucity of N regions in fetal alpha/beta T cells would create a fetal TCR-alpha/beta repertoire that would be quite different from, and smaller than, the adult repertoire. The lack of N regions might be predicted to limit the range of affinities of TCR-MHC + peptide interactions, which may have important consequences for positive and negative selection of fetal and newborn T cells.