Colony-stimulating factor-1 (CSF-1) stimulates motility and cytoplasmic spreading in mature osteoclasts. Therefore, we examined the cellular events and intracellular signaling pathways that accompany CSF-1-induced spreading in normal osteoclasts. To explore the role c-src plays in these processes, we also studied osteoclasts prepared from animals with targeted disruption of the src gene. In normal osteoclasts, CSF-1 treatment induces rapid cytoplasmic spreading, with redistribution of F-actin from a well-delineated central attachment ring to the periphery of the cell. CSF-1 increases membrane phosphotyrosine staining in osteoclasts and induces the phosphorylation of several cellular proteins in cultured, osteoclast-like cells, including c-fms, c-src, and an 85-kD Grb2-binding protein. Src kinase activity is increased threefold after CSF-1 treatment. In src- cells, no attachment ring is present, and CSF-1 fails to induce spreading or a change in the pattern of F-actin distribution. Although c-fms becomes phosphorylated after CSF-1 treatment, the 85-kD protein is significantly less phosphorylated in src- osteoclast-like cells. These results indicate that c-src is critical for the normal cytoskeletal architecture of the osteoclast, and, in its absence, the spreading response induced by CSF-1 is abrogated, and downstream signaling from c-fms is altered.