Signaling pathways mediated by tyrosine phosphorylation and dephosphorylation have been reported to be involved in the regulation of cytoskeletal organization in osteoclasts, the principal cells responsible for bone resorption. We examined the effects of tiludronate [(4-chlorophenyl)thiomethylene bisphosphonate] on the cytoskeleton and the balance of phosphotyrosine levels in osteoclast-like multinucleated cells (OCLs) formed in cocultures of mouse osteoblastic cells and bone marrow cells. When OCLs were placed on plastic dishes in the presence of 10% fetal bovine serum, they formed a ringed structure of F-actin dots (actin ring) within 2 h. Tiludronate did not inhibit the process of actin ring formation, but it disrupted preformed actin rings in a time- and a dose-dependent manner. Western blot analysis using an antiphosphotyrosine antibody revealed that tyrosine phosphorylation of certain proteins in OCLs was stimulated by tiludronate added to the purified OCLs. Tyrosine kinase activity of the p60c-src immunoprecipitated from cell lysates of the purified OCLs was not affected by tiludronate directly added to the kinase assay. OCL lysates stimulated dephosphorylation of tyrosine-phosphorylated substrates such as phosphoneuroprotein 14 and epidermal growth factor receptors. Like sodium orthovanadate, an inhibitor of protein tyrosine phosphatases, tiludronate dose-dependently inhibited tyrosine dephosphorylation of those substrates induced by OCL lysates. These findings suggest that tiludronate disrupts the preformed actin rings and suppresses bone-resorbing activity by inhibiting protein tyrosine phosphatases in osteoclasts.