We are studying the molecular events of T cell development during the first trimester, which is the period of initial colonization of the thymus by T cell precursors and subsequent thymus morphogenesis into defined cortical and medullary areas. We have previously shown that reading frame (RF) usage and functional diversification is developmentally regulated, with virtually all TCRB DI mRNA transcripts using a single RF at 8 weeks of gestational age, tapering to 50% by adult life. To determine whether RF bias is due to selection at the transcriptional or cellular level, we used PCR to create genomic libraries of thymic TCRB DJ rearrangements at 8, 11 and 16 weeks of gestational age, and from an adult thymus. 14S clones were randomly picked and sequenced to determine the extent of junctional diversification and D-region RF usage. We found that the extent of N-region addition and nucleotide deletion at the gene segment ends is reduced during fetal life in genomic rearrangements. In addition, RF usage appears to be stochastic at 8,11, and 16 weeks of gestational age. Similar results were obtained at 8 weeks of gestational age in a second thymus in which both RNA and DNA was isolated and used to create libraries of TCRB DI transcripts or rearrangements. Surprisingly, there was a marked preference (68.2%of unique sequences) for reading frame one in genomic DJ joints from the adult thymus. We conclude that genomic Dlsl-JBlsl rearrangements in reading frame one are preferentially transcribed during fetal life.