We have investigated the nature of immediate cell signals produced by occupancy of the chicken osteoclast alpha v beta 3 integrin. Synthetic osteopontin and peptides from the osteopontin and bone sialoprotein sequences containing Arg-Gly-Asp stimulated immediate reductions in osteoclast cytosolic Ca2+. The changes in cytosolic Ca2+ required the Arg-Gly-Asp sequence and were blocked by a monoclonal antibody to the alpha v beta 3 integrin, LM609. Osteoclast stimulation by the proteins through the integrin did not require immobilization since soluble peptides produced changes in cytosolic Ca2+ and inhibited osteoclast binding to bone particles and bone resorption. The decrease in cytosolic Ca2+ stimulated by osteopontin and related peptides appeared to be due to activation of a plasma membrane Ca(2+)-ATPase by calmodulin. Thus, the data suggest that ligand binding to the osteoclast alpha v beta 3 integrin results in calmodulin-dependent reduction in cytosolic Ca2+ which participates in regulation of osteoclast function.