Zinc has been shown to have an inhibitory effect on osteoclastic bone resorption in vitro. This study was undertaken to determine whether the inhibitory action of zinc on osteoclastogenesis is related to receptor activator of NF-kappaB ligand (RANKL), which plays a pivotal role in differentiation from pre-osteoclasts to osteoclasts. Mouse marrow cells were cultured for 3 days in alpha-minimal essential medium containing lipopolysaccharide (LPS), tumor necrosis factor-alpha (TNFalpha), or RANKL, which stimulates osteoclastogenesis; then zinc sulfate was added to the culture medium containing each osteoclastogenesis-stimulating factor, and the cells were further incubated for 4 days. Osteoclast-like cell formation was estimated by staining for tartrate-resistant acid phosphatase (TRACP), a marker enzyme of osteoclasts. The presence of LPS (10 micro g/ml), TNFalpha (10 ng/ml), or RANKL (10 or 20 ng/ml) with M-CSF (10 or 20 ng/ml) induced a remarkable increase in osteoclast-like multinucleated cells (MNCs). The stimulatory effect of LPS was not significantly altered by the addition of zinc sulfate (10(-6)-10(-4) M). Meanwhile, TNFalpha- or RANKL-induced osteoclast-like cell formation was significantly inhibited in the presence of zinc sulfate (10(-6)-10(-4) M). The effect of zinc sulfate (10(-4) M) in inhibiting RANKL-induced osteoclast-like cell formation was completely abolished in the presence of cycloheximide (10(-7) M), an inhibitor of translation in protein synthesis, or 5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole (DRB; 10(-6) M), an inhibitor of transcription. These results suggest that the inhibitory action of zinc on osteoclastogenesis is partly due to suppressing signaling pathway which is related to RANKL stimulation in osteoclast development.