Terminal deoxynucleotidyl transferase (TdT) expression is controlled at the transcriptional level, however, the TdT core promoter combining D, D', an initiator (Inr) and downstream basal elements (DBE) does not recapitulate the whole complex regulation of TdT expression. We hypothesized that important cis-regulatory elements of the gene are located outside of the TdT promoter. In an attempt to identify these elements, we performed DNase I hypersensitivity assays over 24kb including a 10kb region located upstream of the transcription start site (+1) and a 14kb region spanning exons and introns I to VI. Hypersensitive sites (HS) HS1 and HS2 were localized 8.5 and 8kb upstream of the transcription start site, respectively, and were exclusively detected in TdT+ cell types. HS3, HS4 and HS5 were mapped at positions -7, -3.4 and -3kb, respectively, and detected in both TdT negative and positive cells. HS6, HS7 and HS8 were detected immediately upstream of the TdT promoter. HS10 and HS11 were localized in the first and third intron of the gene. Luciferase reporter assays revealed that HS1, HS2 and HS3 synergize with the TdT promoter to activate transcription in a TdT+ pre-T cell line but not in a TdT+ pro-B cell line. In summary novel cis-regulatory elements have been identified in the 5' region of the TdT locus that synergize with the promoter to activate gene expression and our results suggest these elements may be more active in T cells.