The TCR delta- and alpha-chain genes lie in a single complex locus, the TCRalpha/delta locus. TCRdelta-chain genes are assembled in CD4(-)CD8(-) (double negative (DN)) thymocytes and TCRalpha-chain genes are assembled in CD4(+)CD8(+) (double positive) thymocytes due, in part, to the developmental stage-specific activities of the TCRdelta and TCRalpha enhancers (Edelta and Ealpha), respectively. Edelta functions with TCRdelta promoters to mediate TCRdelta-chain gene assembly in DN thymocytes. However, Edelta is unable to function with TCRalpha promoters such as the TEA promoter to drive TCRalpha-chain gene assembly in these cells. This is important, because the premature assembly of TCRalpha-chain genes in DN thymocytes would disrupt alphabeta and gammadelta T cell development. The basis for TEA inactivity in DN thymocytes is unclear, because Edelta can activate the Vdelta5 gene segment promoter that lies only 4 kb upstream of TEA promoter. In this study, we use gene targeting to construct a modified TCRalpha/delta locus (TCRalpha/delta(5DeltaT)) in which the TEA promoter lies in the same location as the Vdelta5 gene segment on the wild-type TCRalpha/delta 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 Vdelta5 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 Edelta.