TCRbeta chain repertoire of peripheral alphabeta T cells is generated through the stepwise assembly and subsequent selection of TCRbeta V region exons during thymocyte development. To evaluate the influence of a two-step recombination process on Vbeta rearrangement and selection, we generated mice with a preassembled Dbeta1Jbeta1.1 complex on the Jbeta1(omega) allele, an endogenous TCRbeta allele that lacks the Dbeta2-Jbeta2 cluster, creating the Jbeta1(DJbeta) allele. As compared with Jbeta1(omega/omega) mice, both Jbeta1(DJbeta/omega) and Jbeta1(DJbeta/DJbeta) mice exhibited grossly normal thymocyte development and TCRbeta allelic exclusion. In addition, Vbeta rearrangements on Jbeta1(DJbeta) and Jbeta1(omega) alleles were similarly regulated by TCRbeta-mediated feedback regulation. However, in-frame VbetaDJbeta rearrangements were present at a higher level on the Jbeta1(DJbeta) alleles of Jbeta1(DJbeta/omega) alphabeta T cell hybridomas, as compared with on the Jbeta1(omega) alleles. This bias was most likely due to both an increased frequency of Vbeta-to-DJbeta rearrangements on Jbeta1(DJbeta) alleles and a preferential selection of cells with in-frame VbetaDJbeta exons assembled on Jbeta1(DJbeta) alleles during the development of Jbeta1(DJbeta/omega) alphabeta T cells. Consistent with the differential selection of in-frame VbetaDJbeta rearrangements on Jbeta1(DJbeta) alleles, the Vbeta repertoire of alphabeta T cells was significantly altered during alphabeta TCR selection in Jbeta1(DJbeta/omega) and Jbeta1(DJbeta/DJbeta) mice, as compared with in Jbeta1(omega/omega) mice. Our data indicate that the diversity of DJbeta complexes assembled during thymocyte development influences TCRbeta chain selection and peripheral Vbeta repertoire.