CD6 is a lymphocyte surface receptor involved in lymphocyte activation and differentiation processes that is constitutively expressed on developing and mature T cells and on the B1a cells. To define the molecular basis for the tissue-specific expression of CD6 we have identified the transcription factors that control the activity of the proximal regulatory region of the human CD6 gene. The TATA-less CD6 promoter contains multiple transcriptional start sites, and its preferential activity in human T lymphocytes is dependent on RUNX- and Ets-binding sites located within a highly conserved region. RUNX and Ets-1 factors transactivated the CD6 promoter through recognition of the -215 and -230 binding sites, respectively. Chromatin immunoprecipitation assays revealed that RUNX1 constitutively occupies the CD6 promoter in vivo, and knockdown experiments demonstrated that the steady-state level of CD6 mRNA is dependent on the expression of RUNX1, RUNX3 and Ets-1 transcription factors. Therefore, RUNX1/3 and Ets1 control the expression of CD6 in human T lymphocytes, thus expanding the range of T-cell specific and developmentally regulated lymphocyte gene targets involved in T-cell activation and differentiation.