Transforming growth factor beta 1 (TGFB1) is a cytokine involved in the development of both acute and late cutaneous radiation syndromes. We previously demonstrated that ionizing radiation induces TGFB1 expression in vivo in pig skin within a few hours. The purpose of the present study was to develop an in vitro human model to identify the mechanisms of this early activation. Accordingly, human HaCaT keratinocytes were irradiated with a single dose of 20 Gy. First, radiation-induced TGFB1 overexpression was checked at both the transcriptional and transductional levels in HaCaT cells. Then electrophoretic mobility shift assays (EMSA) and transient transfection with various TGFB1 promoter constructs were used to identify the sequences involved in regulating this promoter. EMSA analysis showed the induction of nuclear protein binding activity by gamma irradiation to the -365 AP1 sequence (TGTCTCA), suggesting the involvement of AP1 sequences in the regulation of TGFB1 transcription. In gene reporter assays, maximal TGFB1 promoter activation was found for the longest construct, which contains two AP1 sequences. However, assays with constructs including deletions showed that these two AP1 sequences were not sufficient to confer TGFB1 inducibility. These results showed for the first time, to our knowledge, that transcriptional regulation is involved in radiation-induced activation of TGFB1 gene expression.