Multinucleated osteoclasts from rabbit long bone, 1-6 days in culture, respond to mechanical perturbation with a transient increase of intracellular calcium concentration ([Ca2+]i), as measured with the fluorescent indicator fluo-3 on a confocal laser scanning microscope. In experiments with different extracellular calcium concentrations (from 11.8 mM to calcium-free), the incidence, the magnitude, and the duration of [Ca2+]i responses decreases with decreasing bathing [Ca2+]. Following mechanical perturbation, a thapsigargin-induced [Ca2+]i response has a lower magnitude than the thapsigargin-induced response without mechanical perturbation. In thapsigargin-pretreated osteoclasts the mechanical perturbation-induced rise in [Ca2+]i is larger and longer than in control cells. Ni2+ inhibits the incidence and decreases both the magnitude and the duration of the responses, while nifedipine, verapamil, and Gd3+ have no effect. These measurements show that rabbit osteoclasts transduce a mechanical perturbation of the cell membrane into a [Ca2+]i signal via both a calcium influx and an internal calcium release.