The TCR δ- and α-chain genes lie in a single complex locus, the TCRα/δ locus. TCRδ-chain genes are assembled in CD4 -CD8- (double negative (DN)) thymocytes and TCRα-chain genes are assembled in CD4+CD8+ (double positive) thymocytes due, in part, to the developmental stage-specific activities of the TCRδ and TCRα enhancers (Eδ and Eα), respectively. Eδ functions with TCRδ promoters to mediate TCRδ-chain gene assembly in DN thymocytes. However, Eδ is unable to function with TCRα promoters such as the TEA promoter to drive TCRα-chain gene assembly in these cells. This is important, because the premature assembly of TCRα-chain genes in DN thymocytes would disrupt αβ and γδ T cell development. The basis for TEA inactivity in DN thymocytes is unclear, because Eδ can activate the Vδ5 gene segment promoter that lies only 4 kb upstream of TEA promoter. In this study, we use gene targeting to construct a modified TCRα/δ locus (TCRα/δ5ΔT) in which the TEA promoter lies in the same location as the Vδ5 gene segment on the wild-type TCRα/δ allele. Remarkably, the TEA promoter on this allele exhibits normal developmental stage-specific regulation, being active in double positive thymocytes but not in DN thymocytes as is the case with the Vδ5 promoter. Thus, the inactivity of the TEA promoter in DN thymocytes is due primarily to intrinsic developmental stage-specific features of the promoter itself and not to its location relative to other cis-acting elements in the locus, such as Eδ. Copyright © 2007 by The American Association of Immunologists, Inc.