During fetal life, both Ig and TCR-γδ repertoires are enriched for a specific set of Ag receptors with limited diversity. In order to test the hypothesis that diversification of the human TCR-β repertoire also follows a developmental program, we examined TCR-β DJ and VDJ transcripts from fetal and adult thymi. A consistent bias for Dβ1.1 and Jβ1.1 was present in DJ transcripts amplified from 8-wk gestation thymi. Although it is possible to splice and translate Dβ1.1 in all three reading frames, 8-wk gestation fetal DJ transcripts were enriched for Dβ1.1 spliced to Jβ1.1 in reading frame one. Preference for Dβ1.1 reading frame one and for use of Jβ1.1 dwindled with increasing gestational age. Reading frame bias was not affected by the insertion of non-germ-line-encoded nucleotides (N regions) or by the site of gene splicing. In contrast, choice of reading frame and use of Jβ1.1 appeared random in VDJ transcripts from the same 8-wk gestation and adult thymic samples. Among the occasional VDJ transcripts that contained Dβ1.1- Jβ1.1 rearrangements, use of Dβ reading frame one was rare. Up to 75% of DJ and VDJ transcripts at 8-wk gestation lacked N regions. Both the percentage of DJ and VDJ transcripts with N regions and the average number of inserted nucleotides per transcript increased with gestational age. Primarily as a result of enhanced N region addition, both the length and sequence diversity of the VDJ junctions increased markedly from 8-wk gestation to adult life with a concomitant increase in the potential size of the TCR-αβ repertoire. Terminal deoxynucleotidyl-transferase (TdT), the enzyme associated with N region addition, has not been detected in thymus before 19 wk of gestation. However, we were able to amplify TdT mRNA from all fetal thymi examined. This suggests that TdT may be expressed as early as 8 wk gestation, although at low levels. The early fetal thymus appears enriched for a population of thymocytes that produce minimal quantities of TdT, express a special set of TCR-β DJ transcripts, and generate a TCR-β repertoire with limited diversity. These findings support the hypothesis that limitations in the diversity of the TCR repertoire imposed at the time of gene rearrangement contribute to the immaturity of the fetal immune response.