The bisphosphonates 4-amino-1-hydroxybutylidene-1,1-bisphosphonic acid (ABP), 3-amino-1,1-hydroxypropane-1,1-diphosphonic acid (APD), and 1-hydroxyethane-1,1-diphosphonic acid (EHDP) were compared for their ability to inhibit the osteoclastic resorption of bone in culture. This was achieved by measuring the effect of bisphosphonate concentration on the number of resorption pits formed and the total area of resorption. During this analysis, a new application of reflected polarized light microscopy was developed that has advantages over other microscopy techniques, including scanning electron microscopy (SEM), as applied to the analysis of resorbed bone surfaces. Based on area analysis, the bisphosphonates were effective for the range 10(-7)-10(-8) M, with ABP about two to five times more effective than EHDP or APD. Similar data were obtained by counting the number of resorption pits but with EC50 10 times higher. At lower concentrations (10(-9) M), bisphosphonates may enhance the formation of resorption pits. APD was found to be more toxic (10 times) than ABP or EHDP to osteoclasts and mononuclear cells, but toxic concentrations were at least 10(2) times higher than the resorption EC50. These data plus immunofluorescence, SEM, and transient incubation experiments show that it is the bisphosphonate-bone complex that directly inhibits the excavation of resorption pits by mature osteoclasts. The mechanism of action does not appear to require inhibition of osteoclast differentiation or toxic elimination of osteoclasts. Bisphosphonates, however, subtly affect the cytoskeleton of osteoclasts.