Diverse receptors, including Fcgamma receptors and beta(2) integrins (complement receptor-3 [CR3], CD11b/CD18), have been implicated in phagocytosis, but their distinct roles and interactions with other receptors in particle engulfment are not well defined. CD44, a transmembrane adhesion molecule involved in binding and metabolism of hyaluronan, may have additional functions in regulation of inflammation and phagocytosis. We have recently reported that CD44 is a fully competent phagocytic receptor that is able to trigger ingestion of large particles by macrophages. Here, we investigated the role of coreceptors and intracellular signaling pathways in modulation of CD44-mediated phagocytosis. Using biotinylated erythrocytes coated with specific antibodies (anti-CD44-coated erythrocytes [Ebabs]) as the phagocytic prey, we determined that CD44-mediated phagocytosis is reduced by 45% by a blocking CD11b antibody. Further, CD44-mediated phagocytosis was substantially (42%) reduced in CD18-null mice. Immunofluorescence microscopy revealed that CD11b is recruited to the phagocytic cup. The mechanism of integrin activation and mobilization involved activation of the GTPase Rap1. CD44-mediated phagocytosis was also sensitive to the extracellular concentration of the divalent cation Mg(2+) but not Ca(2+). In addition, buffering of intracellular Ca(2+) did not affect CD44-mediated phagocytosis. Taken together, these data suggest that CD44 stimulation induces inside-out activation of CR3 through the GTPase Rap1.