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Deparaffinized sections of fixed decalcified radii of 2- to 3-day-old rats were incubated in either salmon calcitonin (sCT; 0.1–1.0 MRC U/ml) or in hormone-solvent and then stained for CT by the peroxidase-antiperoxidase method using rabbit antiserum to sCT and 3,3′-diaminobenzidine-H2O2. The sCT-treated radii showed intense brown staining over the cytoplasm of all identifiable osteoclasts and over the osteoblastic layer of the periosteum. Similar intensity of staining was observed in some osteocytes and some mononuclear cells in the marrow spaces. Striated muscle fibers attached to the radii and erythrocytes were also stained. No staining was observed in the chondrocytes, the osteogenic layer of the periosteum, most osteocytes, any of the endosteal osteoblasts, and the majority of mononuclear cells in the marrow spaces. Cell types in sections of bone that stained strongly after sCT incubation also showed faint staining after incubation with CT-solvent. This observation could be due to the presence of endogenous rat CT. In support of this view, binding of sCT antibody to rat CT was also demonstrated by the staining of CT-containing C cells of normal rat thyroid and the lack of staining of thyroid sections obtained from hypercalcemic rats 4 days after continuous infusion (40 mug/24 h) of parathyroid hormone. Furthermore, human CT, which is immunologically similar to rCT, was found to cross-react with antiserum to sCT by RIA. When sections of bone or thyroid were incubated in normal rabbit serum or when peroxidaseantiperoxidase or antibody to rabbit immunoglobulin G was omitted, no brown staining was observed. This study demonstrates that binding of sCT to bone tissue can be localized in the following cells: all osteoclasts, the osteoblastic layer of the periosteum, some mononuclear cells, and some osteocytes.