Interleukin-9 (IL-9) is a multifunctional cytokine that not only has roles in immune and inflammatory responses but also is involved in growth-promoting and anti-apoptotic activities in multiple transformed cell lines, which suggests a potential role in tumorigenesis. Over-expression of the receptor of IL-9 (IL-9R) occurs in several types of human leukemias and in radiation-induced mouse T-cell lymphoma (TL). The molecular mechanism that regulates transcription of the IL-9R gene (Il9r) during leukemogenesis is, however, not well understood. Using a mouse TL cell line that has high expression of Il9r, we sought to dissect its promoter structure. Here we show that the active promoter for Il9r is located in the 5'-flanking AT-rich region. Chromatin immunoprecipitation showed the opening of chromatin structure of the promoter region coupled with nucleolin binding in vivo. Immunohistochemical analysis confirmed the increased localization of nucleolin in the nuclei of TL cells. These data indicate that increased expression of Il9r is associated with an increased binding of nucleolin, coupled with chromatin opening, to an AT-rich region in the 5'-flanking region of Il9r in TL cells.