Abstract Feedback inhibition of V(D)J recombination enforces Ag receptor allelic exclusion in mammalian lymphocytes. Yet, in-frame VβDJβ exons can assemble on both alleles in human and mouse αβ T lineage cells. To elucidate mechanisms that enforce TCRβ allelic exclusion in such cells, we analyzed Vβ expression and rearrangement in mice containing a functional Vβ14DJβ1.5Cβ1 gene (Vβ14NT) and/or Vβ8.2DJβ1.1Cβ1 transgene (Vβ8Tg). The majority of Vβ14NT and Vβ8Tg αβ T lineage cells expressed only Vβ14+ or Vβ8+ TCRβ-chains, respectively, and lacked Vβ rearrangements on wild-type TCRβ loci. However, endogenous Vβ rearrangements and αβ T lineage cells expressing endogenous Vβs from wild-type alleles alone or with the prerearranged Vβ in cell surface TCRβ-chains were observed in Vβ14NT and Vβ8Tg mice. Although nearly all Vβ8Tg:Vβ14NT thymocytes and splenic αβ T cells expressed Vβ8+ TCRβ-chains, only half of these lymphocytes expressed Vβ14+ TCRβ-chains, even though similar steady-state levels of Vβ14NT mRNA were expressed in Vβ8+Vβ14+ and Vβ8+Vβ14− populations. Our data demonstrated that posttranscriptional silencing of functionally assembled endogenous VβDJβCβ genes can enforce TCRβ allelic exclusion and reveal another mechanism that contributes to the development of lymphocytes with monospecific Ag receptors.