The osteoclastic cytoskeleton has been demonstrated to be composed of microfilaments. Osteoclastic multinucleated cells were suspended on dentine slices and cultured for 24 hours in the presence or absence of cytochalasin D (CD), a specific and potent inhibitor of actin filament elongation to determine the role of this cytoskeleton. Cultured cells and co-cultured dentine slices were examined ultrastructurally. Unlike those in control cultures without CD, osteoclasts in CD-treated cultures became spherical in shape and lacked microvilli on their basolateral cell surfaces. Most importantly, CD treatment induced a complete disappearance of the ruffled border-clear zone complexes in osteoclasts, which resulted in loss of osteoclast-cytoplasmic polarity. Morphometric analysis of backscattered electron micrographs of co-cultured dentine slices revealed that CD treatment strongly inhibited the formation of resorption lacunae in a dose-dependent manner. These results suggest that the cytoarchitecture, as well as the bone-resorbing function, of the osteoclast is highly regulated by the F-actin-containing microfilamentous cytoskeleton in the ruffled border-clear zone complex.