The isolated osteoclast bone resorption assay has proved to be a useful means of examining the response of mammalian and avian osteoclasts to a variety of stimuli. The assay has traditionally been performed over a period of 24 hours. By extending the duration of the osteoclast bone resorption assay, we have been able to assess the long-term effects of carboxyl-terminal parathyroid hormone-related protein (hPTHrP[107-139]), salmon calcitonin (sCT) and hPTH[1-34] on bone resorption and TRACP-positive osteoclast-like cell number. We found that, in control cultures over a period of up to 144 hours, the osteoclast-like cells not only remained viable but their numbers also increased. The number of mononucleated and multinucleated osteoclast-like cells doubled in the first 48 hours before stabilizing over the remainder of the incubation period. Osteoblasts also proliferated, resulting in a resorption response to hPTH[1-34] being evident from 48 hours onward. hPTHrP]107-139] persistently inhibited basal and PTH-stimulated bone resorption for at least 96-144 hours, whereas "escape" from the inhibitory effect of sCT was seen after 48-72 hours. Decreased numbers of both mononucleated and multinucleated TRACP-positive osteoclast-like cells were seen by 48 hours in cultures treated with sCT. In contrast, hPTHrP[107-139] reduced the number of mononuclear TRACP-positive cells with only a late effect on multinucleated cells. Furthermore, the increased number of osteoclast-like cells seen in response to hPTH[1-34] was inhibited by carboxyl-terminal PTHrP. In summary, this study indicates that the extended bone resorption assay system is a complex one where both osteoclastic resorption and osteoclast maturation are evident. Using this system, we have shown that hPTHrP[107-139] acts as a potent long-term inhibitor of osteoclastic bone resorption, without evidence of escape from its effect. Its action to reduce the number of mononucleated osteoclast-like cells suggests that it affects several aspects of osteoclast activity.