Hypoxia-induced mitogenic factor (HIMF) possesses mitogenic, vasoconstrictive, angiogenic, and antiapoptotic effects. While HIMF is known to be expressed in developmental mouse lung, its gene expression regulation during this period is completely unknown. Genomic sequencing of HIMF gene has shown that there is an Ets-1 binding site in its 5'-promoter-region. To test the hypothesis that Ets-1 protein expressed in developing mouse lung may participate in the process of HIMF gene expression regulation via direct involvement or facilitation, we characterized the proximal promoter of HIMF gene (409-bp long fragment that includes the -347-bp promoter region from transcription start site), and investigated HIMF and Ets-1 expression with western blot and immunohistochemistry, electrophoretic mobility shift assay (EMSA) for Ets-1, HIMF promoter luciferase reporter gene assays, and chromatin-immunoprecipitation (CHIP). Western blots revealed that both Ets-1 and HIMF proteins were developmentally expressed in the lung. Immunohistochemical localization revealed Ets-1 signals in the nucleus of HIMF-expressing airway epithelial cells and alveolar type II cells, whereas HIMF was localized in cytoplasm. Presence of Ets-1 protein within E16, E20, and adult lung nuclear extract was demonstrated by EMSA. Co-transfection of Ets-1 expression plasmid with HIMF promoter construct increased luciferase activity in NIH3T3 cells, but mutation or deletion of Ets-1 site eliminated HIMF promoter luciferase activity. CHIP with anti-Ets-1 antibody revealed Ets-1 binding to HIMF promoter region in E20 and P1 but not in E15 lung. We conclude that Ets-1 participates in the process of HIMF gene expression and Ets-1-mediated HIMF expression may play an important role in lung development and maturation.