Ultrastructural enzyme and immunocytochemical studies have made great contributions to clarifying intriguing questions as to the actual role of osteoclastic ruffled borders in bone resorption. In the present study, vacuolar-type H(+)-ATPase and cysteine-proteinase (cathepsin) were localized in osteoclasts by means of light and electron microscopic immunocytochemistry. The specific immunoreactivity of vacuolar-type H(+)-ATPase was detected along the ruffled border membranes, associated pale vacuoles, and cisterns of the rough-surfaced endoplasmic reticulum of osteoclasts. Anti-cathepsin B immunoreaction occurred in Golgi vesicles, lysosomes, pale vesicles and vacuoles, and the extracellular canals of ruffled borders of osteoclasts. The resorbing bone surfaces were also immunoreactive for anti-cathepsin B. In a coculture system of osteoclasts with devitalized dentine slices, a specific H(+)-ATPase inhibitor (bafilomycin A1) markedly reduced both demineralized areas and resorption lacuna formation on the dentine slices. On the other hand, the cathepsin inhibitor, E-64, inhibited only resorption lacuna formation but had no effect on demineralization of the dentine slices. These results suggest that H(+)-ATPase and cathepsins in osteoclasts are involved, respectively, in the extracellular solubilization of apatite crystals and subsequent degradation of bone matrix and that the ruffled border-clear zone complex of osteoclasts is the main site of cell-matrix interactions during bone resorption processes.