Overexpression of the neutrophil gelatinase-associated lipocalin (NGAL) gene is related to malignant transformation of human immortalized esophageal epithelial cell induced by 12-O-tetradecanoyl phorbol 13-acetate (TPA). However, the mechanisms of the transcriptional activation of this gene remain unclear. The recent study indicated that there might be TPA response elements in the 5′flanking promoter region and the sequences surrounding it. In order to locate the position of TPA response elements, the NGAL fragments for －152～+84, －140～+84, －78～+84, －59～+84, －50～+84, －41～+84, －37～+84, －29～+84 and －10～+84 were obtained from esophageal cancer cells by PCR and nested deletion and cloned into pGL3-Basic (pGLB), pGL3-Enhancer (pGLE) and pGL3-promoter (pGLP) vector which are luciferase reportors, respectively. The constructed recombinant expression plasmids pGLB-152, pGLP-152, pGLE-152, pGLB-140, pGLB-78, pGLB-59, pGLB-50, pGLB-41, pGLB-37, pGLB-29 and pGLB-10 were adopted to cotransfect with pRL-TK into EC109 esophageal cancer cells stimulated with TPA (5 μg/L) respectively or not. Relative luciferase activity of whole cell extracts from cells was measured with Dual Luciferase Report Gene Assay System (DLR) to verify the position of TPA response elements. Results showed that the TPA response elements of NGAL are located on －152～－60 region and have a strong response to TPA stimulation in the esophageal cancer cells. The analysis by bioinformatics showed that the potential TPA responsive element in －152～－60 position of NGAL is a new sequence. This suggested that there is the structural character responding to TPA induction in the gene. These results will help to clarify the molecular mechanisms of NGAL overexpression in malignant transformation of human immortalized esophageal epithelial cell induced by TPA.