V(D)J recombination and transcription within the TCRα/δ locus are regulated by three characterized cis-acting elements: the TCRδ enhancer (Eδ), TCRα enhancer (Eα), and T early α (TEA) promoter. Analysis of enhancer and promoter occupancy and function in developing thymocytes in vivo indicates Eδ and Eα to be developmental-stage-specific enhancers, with Eδ 'on' and Eα 'off' in double-negative III thymocytes and Eδ 'off' and Eα 'on' in double-positive thymocytes. Eδ downregulation reflects a loss of occupancy. Surprisingly, Eα and TEA are extensively occupied even prior to activation. TCRδ downregulation in double-positive thymocytes depends on two events, Eδ inactivation and removal of TCRδ from the influence of Eα by chromosomal excision.