Receptor activator of nuclear factor-κB ligand (RANKL)-induced signals play critical roles in osteoclast differentiation and function. SB203580, an inhibitor of p38 MAPK, blocked osteoclast formation induced by 1α,25-dihydroxyvitamin D₃ and prostaglandin E₂ in cocultures of mouse osteoblasts and bone marrow cells. Nevertheless, SB203580 showed no inhibitory effect on RANKL expression in osteoblasts treated with 1α,25-dihydroxyvitamin D₃ and prostaglandin E₂. RANKL-induced osteoclastogenesis in bone marrow cultures was inhibited by SB203580, suggesting a direct effect of SB203580 on osteoclast precursors, but not on osteoblasts, in osteoclast differentiation. However, SB203580 inhibited neither the survival nor dentine-resorption activity of osteoclasts induced by RANKL. Lipopolysaccharide (LPS), IL-1, and TNFα all stimulated the survival of osteoclasts, which was not inhibited by SB203580. Phosphorylation of p38 MAPK was induced by RANKL, IL-1, TNFα, and LPS in osteoclast precursors but not in osteoclasts. LPS stimulated phosphorylation of MAPK kinase 3/6 and ATF2, upstream and downstream signals of p38 MAPK, respectively, in osteoclast precursors but not in osteoclasts. Nevertheless, LPS induced degradation of IκB and phosphorylation of ERK in osteoclasts as well as in osteoclast precursors. These results suggest that osteoclast function is induced through a mechanism independent of p38 MAPK-mediated signaling.