Osteoblasts or bone marrow stromal cells are required as supporting cells for the in vitro differentiation of osteoclasts from their progenitor cells. Soluble receptor activator of nuclear factor-κB ligand (RANKL) in the presence of macrophage colony-stimulating factor (M-CSF) is capable of replacing the supporting cells in promoting osteoclastogenesis. In the present study, using Balb/c-derived cultures, osteoclast formation in both systems—osteoblast/bone-marrow cell co-cultures and in RANKL-induced osteoclastogenesis—was inhibited by antibody to tumor necrosis factor-α (TNF-α), and was enhanced by the addition of this cytokine. TNF-α itself promoted osteoclastogenesis in the presence of M-CSF. However, even at high concentrations of TNF-α the efficiency of this activity was much lower than the osteoclastogenic activity of RANKL. RANKL increased the level of TNF-α mRNA and induced TNF-α release from osteoclast progenitors. Furthermore, antibody to p55 TNF-α receptors (TNF receptors-1) (but not to p75 TNF-α receptors (TNF receptors-2) inhibited effectively RANKL- (and TNF-α() induced osteoclastogenesis. Anti-TNF receptors-1 antibody failed to inhibit osteoclastogenesis in C57BL/6-derived cultures. Taken together, our data support the hypothesis that in Balb/c, but not in C57BL/6 (strains known to differ in inflammatory responses and cytokine modulation), TNF-α is an autocrine factor in osteoclasts, promoting their differentiation, and mediates, at least in part, RANKL's induction of osteoclastogenesis. © 2001 Wiley-Liss, Inc.