Although tightly linked, the TCR α and δ genes are expressed specifically in T lymphocytes, whereas the Dad1 gene is ubiquitously expressed. Between TCR α and Dad1 are eight DNase I hypersensitive sites (HS). HS1 colocalizes with the TCR α enhancer (E α) and is T cell-specific; HS2,-3,-4,-5, and-6 map downstream of HS1 and are tissue-nonspecific. The region spanning HS2–6 was reported to display chromatin-opening activity and to confer copy number-dependent and integration site-independent transgene expression in transgenic mice. Here, we demonstrate that HS2–6 also displays enhancer-blocking activity, as it can block an enhancer from activating a promoter when located between the two in a chromatin-integrated context, and can do so without repressing either the enhancer or the promoter. Multiple enhancer-blocking elements are arrayed across HS2–6. We show that HS2–6 by itself does not activate transcription in chromatin context, but can synergize with an enhancer when located upstream of an enhancer and promoter. We propose that HS2–6 primarily functions as an insulator or boundary element that may be critical for the autonomous regulation of the TCR α and Dad1 genes.