The coreceptors CD4 and CD8 play a crucial role during thymocyte development and T cell effector function, and their expression is developmentally regulated. To determine the underlying molecular mechanisms of CD8 gene regulation we cloned the murine CD8 gene locus from genomic libraries and analyzed this region for deoxyribonuclease (DNase I) hypersensitive sites (HSS). Here we report, using transgenic mice, deletion analysis of one of the identified clusters of DNase I hypersensitivity, consisting of three DNase I-HSS and located in the intergenic region between the CD8alpha and CD8beta genes. Our data show that at least two of the DNase I-HSS constituting this cluster are individually sufficient to direct CD8alpha or heterologous transgene expression to the mature CD8 single-positive T cell subset and that this expression coincides temporally with the appearance of positively selected T cells.