Although much has been learned recently of the mechanisms that regulate osteoclastic differentiation, much less is known of the means through which their resorptive activity is controlled. We have developed an assay that allows us to measure resorptive activity while minimizing the confounding effects of the test agent on differentiation. In this assay, murine osteoclasts were harvested from plastic substrates and sedimented onto bone slices in MEM with 10% fetal calf serum. The majority excavate the bone surface within a few hours. We found that the regulation of resorption was distinct from that of osteoclastogenesis. Thus, interferons-β and -γ, which strongly suppress, and TGFβ, which potently stimulates osteoclast differentiation, were without effect on resorption, whereas IL-1α, which does not induce osteoclastogenesis, was a strikingly potent stimulus for bone resorption. TNFα and IL-1α were able to replace receptor activator of nuclear factor-κB ligand for stimulation of bone resorption. Protons stimulated bone resorption only in the presence of a resorptive stimulus. PTH, IL-6, and antibodies against osteoclast-associated receptor did not affect bone resorption. Resorption was potently suppressed by 20 mm calcium, 10 μm cyclosporin A, 1 ng/ml calcitonin, and 1 mm dibutyryl cAMP and cGMP. These results show that full functional differentiation of osteoclasts does not require a signal from bone matrix but can occur on plastic and that osteoclastic differentiation and function are regulated by distinct agents.