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% FCS. 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 and , which strongly suppress, and TGF-, which potently stimulates osteoclast differentiation, were without effect on resorption, while IL-1, which does not induce osteoclastogenesis, was a strikingly potent stimulus for bone resorption. TNF- and IL-1 were able to replace RANKL for stimulation of bone resorption. Protons stimulated bone resorption only in the presence of a resorptive stimulus. Neither PTH, IL-6, nor antibodies against osteoclast-associated receptor (OSCAR), affected bone resorption. Resorption was potently suppressed by 20 mM calcium, 10 µM cyclosporin A, 1ng/ml CT, and 1 mM dibutyryl cyclic-AMP and -GMP. 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.