Dichloromethylenebisphosphonate (Cl2MBP, formerly Cl2MDP) inhibits bone resorption in vivo and in vitro. The mechanism by which it inhibits osteoclastic bone resorption has not been established. To investigate this, osteoclasts were isolated from rat long bone and incubated with Cl2MBP (10(-9)-10(-5) M) on bone slices. Bone resorption was assessed as plan area resorbed after 6 and 24 h by scanning electron microscopy. Although Cl2MBP inhibited bone resorption in the first 6 h of culture, inhibition was more marked in the incubation period between 6 and 24 h. This pattern of accelerating inhibition is unlike the pattern we have observed using other resorption-inhibitors, and suggested resorption-mediated osteoclast injury. In keeping with this, we found reduced numbers of osteoclasts, and morphological features of cell injury and degeneration of osteoclasts, after incubation with Cl2MBP on bone slices. Bone seemed to be an essential component in Cl2MBP-mediated injury, since osteoclast numbers and morphology on plastic coverslips were unaffected by the bisphosphonate. Moreover, bone slices preincubated with Cl2MBP showed similar effects on resorption and morphology to cultures in in which osteoclasts on bone were continuously immersed in Cl2MBP. Neither non-resorptive cells (macrophages, UMR 106 cells), nor osteoclasts prevented from resorption by calcitonin, showed evidence of cytotoxicity after incubation on bone slices with Cl2MBP. These results suggest that even relatively high concentrations of Cl2MBP in the fluid phase do not affect osteoclasts, nor does contact with Cl2MBP-coated bone surfaces, but that injury to osteoclasts, and a consequent reduction in bone resorption, occurs when osteoclasts excavate bone surfaces upon which Cl2MBP is adsorbed.