A noncompetitive enzyme immunoassay method (hetero‐two‐site enzyme immunoassay) for salmon calcitonin (SCT) and its usability for the pharmacokinetic study are described. The method in brief proceeds as follows: centrifugal filtration through a polysaccharide membrane to remove plasma proteins, biotinylation, trapping onto an anti‐SCT IgG‐coated polystyrene ball, acid elution, coupling with affinity‐purified anti‐SCT Fab¹‐peroxidase conjugate, final trapping onto streptavidin‐coated polystyrene balls, and measurement of peroxidase activity bound to the balls by fluorometry. The practical detection limit of SCT was 0.1 pg (30 amol)/assay and 2 pg/ml as the assay sample's concentration, which was at least fivefold lower than those previously reported by competitive radioimmunoassays. The application of this method has enabled us to 1) directly estimate the bioavailability of SCT dosed subcutaneously at the therapeutic levels (1.2 and 4.7 μg/kg) for its antiosteoporotic effect as compared to an intravenous dose (1.2 μg/kg) and 2) search for the relationship between blood level and the hypocalcemic activity of SCT. The pharmacokinetic parameters of subcutaneous SCT (1.2 and 4.7 μg/kg) thus estimated were as follows: the area under the blood concentration‐time curve (AUC) = 89 and 550 pg hr/ml, and mean residence time (MRT) = 44 and 65 minutes, respectively, when the AUC for an intravenous SCT (1.2 μg/kg) = 160 pg hr/ml and the MRT = 10 minutes. © 1996 Wiley‐Liss, Inc.