We have isolated the genomic sequence of human interleukin-9 (IL-9) based on its sequence homology with a human IL-9 cDNA isolated from human T-cell leukemia virus (HTLV)-I-transformed T cells by expression cloning. The entire genomic sequence has been determined and the gene consists of five exons and four introns. The human IL-9 gene is mapped to the long arm of human chromosome 5 at band 5q31–32, a region found to be deleted in a number of patients with acquired 5q- abnormalities and hematologic disorders. Several blocks of transcriptional control sequences have been identified at the 5′-flanking region of the human IL-9 gene that may play an important role in the control of IL-9 gene expression. The 5′-regulatory region of the human IL-9 gene also contains sequences identified in the 5′-flanking regions of other cytokine genes mapped to the long arm of human chromosome 5, including IL-3, IL-4, IL-5, and granulocyte-macrophage colony-stimulating factor and other T-cell growth factor genes including IL-2 and IL-6. The IL-9 gene is constitutively expressed in the HTLV-I-transformed human T cells and the expression of IL-9 in these cells can be further induced by 12-O-tetradecanoyl phorbol 13-acetate. Transient transfection analysis using the plasmid containing the 5′-flanking region of IL-9 gene upstream from the firefly luciferase ciferase report gene indicated that the 0.9-kb Smal-Sacl fragment of the IL-9 gene contains sequences required for the constitutive and activated expression of IL- 9 gene in HTLV-I-transformed cells. These results will now allow us to study the regulatory mechanism of IL-9 gene expression in normal and leukemic human T cells.