IL-6, leukemia inhibitory factor (LIF), and oncostatin M (OSM) are IL-6-type cytokines that stimulate osteoclast formation and function. In the present study, the resorptive effects of these agents and their regulation of receptor activator of NF-κB ligand (RANKL), RANK, and osteoprotegerin (OPG) were studied in neonatal mouse calvaria. When tested separately, neither human (h) IL-6 nor the human soluble IL-6R (shIL-6R) stimulated bone resorption, but when hIL-6 and the shIL-6R were combined, significant stimulation of both mineral and matrix release from bone explants was noted. Semiquantitative RT-PCR showed that hIL-6 plus shIL-6R enhanced the expression of RANKL and OPG in calvarial bones, but decreased RANK expression. Human LIF, hOSM, and mouse OSM (mOSM) also stimulated 45 Ca release and enhanced the mRNA expression of RANKL and OPG in mouse calvaria, but had no effect on the expression of RANK. In agreement with the RT-PCR analyses, ELISA measurements showed that both hIL-6 plus shIL-6R and mOSM increased RANKL and OPG proteins. 1, 25-Dihydroxyvitamin D 3 (D 3) also increased the RANKL protein level, but decreased the protein level of OPG. OPG inhibited 45 Ca release stimulated by RANKL, hIL-6 plus shIL-6R, hLIF, hOSM, mOSM, and D 3. An Ab neutralizing mouse gp130 inhibited 45 Ca release induced by hIL-6 plus shIL-6R. These experiments demonstrated stimulation of calvarial bone resorption and regulation of mRNA and protein expression of RANKL and OPG by D 3 and IL-6 family cytokines as well as regulation of RANK expression in preosteoclasts/osteoclasts of mouse calvaria by D 3 and hIL-6 plus shIL-6R.