T cell receptor (TCR) δ and α variable region genes are assembled from germ-line gene segments located in a single chromosomal locus in which TCRδ segments are situated between TCRα segments. The TCRα enhancer (Eα) located at the 3′ end of the TCRα/δ locus functions over a long chromosomal distance to promote TCRα rearrangement and maximal TCRδ expression; whereas the TCRδ enhancer (Eδ) is located among the TCRδ segments and functions with additional element(s) to mediate TCRδ rearrangement. We used gene-targeted mutation to evaluate whether the identity of Eα and the position of Eδ are critical for the developmental stage-specific assembly of TCR δ and α variable region genes. Specific replacement of Eα with Eδ, the core Eα element (EαC), or the Ig heavy chain intronic enhancer (iEμ), all of which promote accessibility in the context of transgenic V(D)J recombination substrates, did not promote a significant level of TCRα rearrangement beyond that observed in the absence of Eα. Therefore, the identity and full complement of Eα-binding sites are critical for promoting accessibility within the TCRα locus. In the absence of the endogenous Eδ element, specific replacement of Eα with Eδ also did not promote TCRδ rearrangement. However, deletion of intervening TCRα/δ locus sequences to restore the inserted Eδ to its normal chromosomal position relative to 5′ sequences rescued TCRδ rearrangement. Therefore, unlike Eα, Eδ lacks ability to function over the large intervening TCRα locus and/or Eδ function requires proximity to additional upstream element(s) to promote TCRδ accessibility.