We developed and validated the first serum enzyme-linked immunosorbent assay for hepcidin, the principal iron-regulatory hormone that has been very difficult to measure. In healthy volunteers, the 5% to 95% range of hepcidin concentrations was 29 to 254 ng/mL in men (n = 65) and 17 to 286 ng/mL in women (n = 49), with median concentrations 112 versus 65 (P < .001). The lower limit of detection was 5 ng/mL. Serum hepcidin concentrations in 24 healthy subjects correlated well with their urinary hepcidin (r = 0.82). Serum hepcidin appropriately correlated with serum ferritin (r = 0.63), reflecting the regulation of both proteins by iron stores. Healthy volunteers showed a diurnal increase of serum hepcidin at noon and 8 pm compared with 8 am, and a transient rise of serum hepcidin in response to iron ingestion. Expected alterations in hepcidin levels were observed in a variety of clinical conditions associated with iron disturbances. Serum hepcidin concentrations were undetectable or low in patients with iron deficiency anemia (ferritin < 10 ng/mL), iron-depleted HFE hemochromatosis, and juvenile hemochromatosis. Serum hepcidin concentrations were high in patients with inflammation (C-reactive protein > 10 mg/dL), multiple myeloma, or chronic kidney disease. The new serum hepcidin enzyme-linked immunosorbent assay yields accurate and reproducible measurements that appropriately reflect physiologic, pathologic, and genetic influences, and is informative about the etiology of iron disorders.