To date there have been few published immunoassays for the important iron regulator hepcidin. This study describes a novel competitive radioimmunoassay (RIA) for the bioactive hepcidin peptide. A rabbit anti-hepcidin polyclonal antibody was produced using synthetic hepcidin radiolabelled with 125I to produce a competitive RIA. Normal patient (n=47) samples were collected and assayed for hepcidin to determine a reference range. Other patient groups collected were ulcerative colitis (UC; n=40), iron deficiency anaemia (IDA; n=15), chronic kidney disease not requiring dialysis (CKD; n=45) and chronic kidney disease requiring dialysis (HCKD; n=94). Detection limit of the assay was determined as 0.6 ng/mL. Intra-assay precision was 5 ng/mL (7.2%) and 50 ng/mL (5.8%), interassay precision was 5 ng/mL (7.6%) and 50 ng/mL (6.7%). Analytical recovery was 98% (5 ng/mL), 94% (10 ng/mL) and 97% (50 ng/mL). The assay was linear up to 200 ng/mL. No demonstrable cross-reactivity with human insulin, glucagon I, angiotensinogen I, beta-defensin 1-4, alpha-defensin-1 and plectasin was observed. There was significant correlation (r=0.96, P < or = 0.0001) between the hepcidin RIA and an established hepcidin SELDI-TOF-MS method. Analysis of the normal human samples gave a reference range of 1.1-55 ng/mL for hepcidin. Further statistical evaluation revealed a significant difference between male and female hepcidin levels. There was significant correlation between hepcidin and ferritin in the control group (r=0.6, P < or = 0.0001). There was also a significant difference between the normal and disease groups (P < or = 0.0001). Healthy volunteers (n=10) showed a diurnal increase in plasma hepcidin at 4.00 pm compared to 8.00 am. A robust and optimised immunoassay for bioactive hepcidin has been produced and the patient sample results obtained further validates the important role of hepcidin in iron regulation, and will allow further investigation of this important peptide and its role in iron homeostasis.