Occupancy of the chicken osteoclast αvβ3 integrin stimulates immediate cell signals. Peptides from osteopontin containing Arg-Gly-Asp 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 LM609, a monoclonal antibody to the αvβ3 integrin. 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 was due to activation of a plasma membrane Ca2+-ATPase. Thus, the data suggest that ligand binding to the osteoclast αvβ3 integrin results in a reduction in cytosolic Ca2+ which participates in regulation of osteoclast function.