Effects of concanamycin B, a specific inhibitor of vacuolar type H^+-ATPase (V-ATPase), and prodigiosin 25-C, which inhibits proton pump activity of V-ATPase without affecting its ATPase activity, on acidification of acidic intracellular granules in osteoclasts and on osteoclastic bone resorption were determined. These two inhibitors suppressed the acidification of the granules as well as bone resorption. Destruxins A, B and B, which are known to inhibit proton pump activity of V-ATPase, did not inhibit acidification of acidic intracellular granules in osteoclasts. However, they inhibited osteoclastic bone resorption by inducing morphological changes of osteoclasts that are not observed in the osteoclasts treated with concanamycin B or prodigiosin 25-C.It is clearly demonstrated that the morphological changes caused by destruxins are attributable to disruption of actin rings in osteoclasts which are essential cytoskeletal structures for bone resorption. The disruptive potency of destruxin E, which contains epoxide moiety in the molecule, was 30 times more evident than that of destruxin A and B, suggesting that the epoxide plays an important role in the expression of biological activities.