The human EBAG9 was previously identified as an estrogen responsive gene using CpG-genomic binding site cloning (Watanate et al, (1998) Mel. Cell. Biol. 18: 442-449). Recently it was revealed that the EBAG9 is identical with RCAS1 which is a cancer cell. surface antigen implicated in immune escape. Here, we isolated and analyzed the 5'-flanking region of human EBAG9 gene. We determined transcription initiation site, which has a homology with an initiator element YYCAYYYY, and found that TATA motif was absent. Deletion analysis of the 5'-flanking region using MCF-7 breast cancer cells indicated that the sequences -86 to -36 containing the ERE had the basal level of promoter activity and the upstream CC-rich region positively regulated the activity. EBAG9 pro meter luciferase reporters containing the ERE could respond to estrogen, and electrophoretic mobility shift assay showed that ER alpha bound to the ERE. Moreover, fluorescent in situ hybridization analysis has shown that the human EBAG9 gene is located at chromosome 8q23 which is frequently amplified in tumors. These findings suggest that the human EBAG9 might be involved in carcinogenesis as an estrogen responsive gene,