Salmon calcitonin (sCT) is widely used therapeutically in the treatment of patients with postmenopausal osteoporosis, Paget's disease, and some forms of hypercalcemia. Preparations of synthetic calcitonin peptides of high purity and reproducibility are now routinely produced and physicochemical methods, particularly reverse-phase high-performance liquid chromatography (RP-HPLC), are replacing the in vivo biological assay for monitoring and calibration. Although the bioassay is no longer required for routine batch control in Europe, calcitonin bioassays are still required in some countries and in the development of new products. Stocks of the Second International Standard (IS) for salmon calcitonin are now depleted and, to replace it with a new calibrant, an international collaborative study was organized in which the aims were to: determine the activity of the candidate sCT by in vivo bioassay in terms of the second IS; assess the stability of the preparation after accelerated thermal degradation; estimate the purity of the ampouled candidate preparation; and determine the sCT content in gravimetric units by HPLC. The HPLC data in terms of ampoule content were in good agreement giving an estimate of 23.1 (coefficient of variation [CV] 3.8%) microg per ampoule. The HPLC chromatograms revealed a small, but detectable, degree of heterogeneity, which possibly occurred during the formulating or ampouling procedures, resulting in a reduction in monocomponent content (purity) from 96% to 92%. The biological activity of the ampoule contents in international units (IU) was calculated from the mass value and the internationally agreed-upon figure of 6000 IU/per mg for the specific activity of salmon calcitonin. This gave a value of 138 IU per ampoule, which was in good agreement with the biological assay estimate (140 IU per ampoule). The preparation of sCT was subsequently adopted as the Third International Standard by the World Health Organization with an assigned content of 138 IU per ampoule.