The actions of 1,25-dihydroxycholecalciferol [1,25-(OH)2D3] and parathormone, both effective bone-resorptive agents in vivo and in vitro, were tested on CT (osteoclast-like) and PT (osteoblast-like) bone cells maintained in culture. Both agents stimulated acid phosphatase activity and hyaluronate synthesis in the CT cells and decreased alkaline phosphatase, citrate decarboxylation, and collagen synthesis in the PT cells. Calcitonin inhibited the changes induced in the CT but not in the PT cells. The activity of 1,25-(OH)2D3 differed from that of parathormone in one key respect: it did not increase cellular cyclic adenosine monophosphate, whereas parathormone did. Prior incubation of the bone cells with 1,25-(OH)2D3 for 6 to 24 hours made the cells refractory to the effect of parathormone on cyclic adenosine monophosphate formation. These data suggest that 1,25-(OH)2D3 and parathormone induce bone resorption by affecting the same cell types (osteoblasts and osteoclasts) although at different cellular sites.