Neutrophil-independent macrophage responses are a prominent part of delayed-type immune and healing processes and depend on T cell-secreted cytokines. An important mediator in this setting is the phosphoprotein osteopontin, whose secretion by activated T cells confers resistance to infection by several intracellular pathogens through recruitment and activation of macrophages. Here, we analyze the structural basis of this activity following cleavage of the phosphoprotein by thrombin into two fragments. An interaction between the C-terminal domain of osteopontin and the receptor CD44 induces macrophage chemotaxis, and engagement of beta(3)-integrin receptors by a nonoverlapping N-terminal osteopontin domain induces cell spreading and subsequent activation. Serine phosphorylation of the osteopontin molecule on specific sites is required for functional interaction with integrin but not CD44 receptors. Thus, in addition to regulation of intracellular enzymes and substrates, phosphorylation also regulates the biological activity of secreted cytokines. These data, taken as a whole, indicate that the activities of distinct osteopontin domains are required to coordinate macrophage migration and activation and may bear on incompletely understood mechanisms of delayed-type hypersensitivity, wound healing, and granulomatous disease.