In order to explore the distribution of hormone-responsive cells in skeletal tissues, we have examined the effects of synthetic bovine parathyroid hormone N-terminal peptide (bPTH 1–34) and salmon calcitonin (sCT) on cyclic AMP levels in periosteum-free rat calvaria, segments of periosteum, and in isolated cells dispersed from each tissue by collagenase digestion. Synthetic bovine PTH increased cyclic AMP levels to a greater degree in calvaria and in isolated bone cells than in the periosteal segments and cells, whereas sCT was more effective in the periosteal than in the bone systems. Primary cultures prepared from bone and periosteal cell populations exhibited progressive increases in their responsiveness to bPTH (1–34) and progressive decreases in responsiveness to sCT. After six days in the culture, bone cells failed to respond to sCT, and sCT did not modify their response to simultaneously added bPTH (1–34). Sixday periosteal cell cultures exhibited residual sCT responsivity and an additive response upon simultaneous exposure to high concentrations of bPTH (1–34) and sCT suggesting separate sites of hormone action. Adenosine, a known stimulator of bone cell adenylyl cyclase, caused a greater increase in periosteal cell than in bone cell cyclic AMP. bPTH (l–34)-responsive cells which enrich periosteum-free bone may be osteoblasts, in view of their histological prominence in this tissue and in the bone cell isolates. Periosteal cells which responded to sCT and to adenosine preferentially are unidentified. Although periosteal segments contained numerous fibroblast-like cells, skin fibroblasts cultured from the same fetuses were sCTinsensitive. Growth in primary culture appears to alter the number of hormone-responsive cells or responsiveness of existing cells to each hormone, or both.