A novel and sensitive noncompetitive enzyme immunoassay (hetero-two-site enzyme immunoassay) for alpha-human atrial natriuretic peptide (alpha-hANP) in plasma, which uses only one monoclonal IgG for the ring structure of alpha-hANP, is described. Plasma was filtered through polysaccharide membrane to separate peptides from proteins. The plasma filtrate was incubated with N-hydroxysuccinimidobiotin to biotinylate alpha-hANP and subsequently with a polystyrene ball coated with monoclonal IgG for the ring structure of alpha-hANP to trap biotinylated alpha-hANP. The polystyrene ball was washed to eliminate unreacted N-hydroxysuccinimidobiotin and other biotinylated substances, and biotinylated alpha-hANP was eluted from the polystyrene ball with HCl. The eluate was neutralized and incubated with horseradish peroxidase-labeled antibody IgG for the ring structure of alpha-hANP and subsequently with two streptavidin-coated polystyrene balls. Peroxidase activity bound to the streptavidin-coated polystyrene balls was assayed by fluorometry. The detection limit of alpha-hANP was 20 amol, and the assay range of plasma alpha-hANP was 0.8-1,200 ng/L using 100 microliters of plasma filtrates corresponding to 75 microliters of plasma. Plasma levels of hANP in healthy subjects were 9.8-21.5 ng/L. These values were significantly lower than those measured by a two-site enzyme immunoassay probably due to the presence of alpha-hANPs lacking some N-terminal amino acids, which were as reactive as alpha-hANP in the two-site enzyme immunoassay but less reactive in the hetero-two-site enzyme immunoassay.