Ureteral obstruction is characterized by decreased renal blood flow that is associated with hypoxia within the kidney. Adrenomedullin (AM) is a peptide hormone with tissue-protective capacity that is stimulated through hypoxia. We tested the hypothesis that ureteral obstruction stimulates expression of AM and hypoxia-inducible factor-1 (HIF-1α) in kidneys. Rats were exposed to bilateral ureteral obstruction (BUO) for 2, 6, 12, and 24 h or sham operation and compared with unilateral obstruction (UUO). AM mRNA expression was measured by quantitative PCR in cortex and outer medulla (C+OM) and inner medulla (IM). AM and HIF-1α protein abundance and localization were determined in rats subjected to 24-h BUO. AM mRNA expression in C+OM increased significantly after 12-h BUO and further increased after 24 h. In IM, AM mRNA expression increased significantly in response to BUO for 6 h and further increased after 24 h. AM peptide abundance was enhanced in C+OM and IM after 24-h BUO. Immunohistochemical labeling of kidneys showed a wider distribution and more intense AM signal in 24-h BUO compared with Sham. In UUO rats, AM mRNA expression increased significantly in IM of the obstructed kidney compared with nonobstructed and Sham kidney whereas AM peptide increased in IM compared with Sham. HIF-1α protein abundance increased significantly in IM after 24-h BUO compared with Sham and HIF-1α immunoreactive protein colocalized with AM. In summary, AM and HIF-1α expression increases in response to ureteral obstruction in agreement with expected oxygen gradients. Hypoxia acting through HIF-1α accumulation may be an important pathway for the renal response to ureteral obstruction.