Recent advances in technique have made it possible to study human osteoclast (OC) formation and activity in vitro. The object of the present study was to determine the effects of alendronate (ALN) on human OCs generated from precursors obtained from standard peripheral blood samples. Peripheral blood mononuclear cells from 14 postmenopausal women were cocultured with ST2 stromal cells on bone slices in the presence of 10(-7) M 1,25-dihydroxyvitamin D3, 10(-8) M dexamethasone, and 25 ng/ml human macrophage colony-stimulating factor. After 21 days, the cultures contained numerous OCs, which were characterized by multinuclearity, the presence of tartrate-resistant acid phosphatase, calcitonin and vitronectin receptors, and the ability to resorb substantial amounts of bone, which was inhibited by calcitonin. The percentage area of bone resorbed per slice was highly correlated (r = 0.89, p < 0.001) with the concentration of Type I collagen cross-linked C-telopeptides (CTx) released into the culture medium. When added to the medium, ALN inhibited bone resorption at concentrations < or =10(-7) M. At 10(-7) M, inhibition was achieved primarily by a reduction in OC activity without a marked effect on OC number. At the highest concentration studied (10(-5) M), both OC number and resorption were profoundly decreased. Overnight preincubation of bone slices in ALN, without further exposure to ALN, resulted in an inhibition of resorption that was similar to that seen when ALN was present in the medium throughout the entire culture period. We conclude that, except at very high concentrations, the predominant mechanism of action of ALN is to inhibit the activity of differentiated human OCs with little or no effect on recruitment. Interaction between the OC and ALN on the bone surface is an important component of the inhibitory mechanism. Measurement of CTx in tissue culture medium is a convenient method for assessment of bone resorption in human OC cultures and offers a number of advantages over morphometric analysis of the bone slice.