The TCRbeta gene enhancer (Ebeta) commands TCRbeta gene expression through the lifespan of T lymphocytes. Genetic and molecular studies have implied that in early thymocytes, Ebeta directs chromatin opening over the Dbeta-Jbeta-Cbeta domains and triggers initial Dbeta-Jbeta recombination. In mature T cells, Ebeta is required for expression of the assembled TCRbeta gene. Whether these separate activities rely on distinct Ebeta regulatory sequences and involve differing modes of activation is unclear. Using gene targeting in mouse embryonic stem cells, we replaced Ebeta by a conserved core fragment (Ebeta169). We found that Ebeta169-carrying alleles were capable of sustaining beta gene expression and the development of mature T cells in homozygous knockin mice. Surprisingly, these procedures and underlying molecular transactions were affected to a wide range of degrees depending on the developmental stage. Early thymocytes barely achieved Dbeta-Jbeta germline transcription and recombination. In contrast, T cells displayed substantial though heterogeneous levels of VDJ-rearranged TCRbeta gene expression. Our results have implications regarding enhancer function in cells of the adaptive immune system and, potentially, TCRbeta gene recombination and allelic exclusion.