The importance of cytoplasmic motifs in differentially regulating SR-A function was demonstrated by deleting the first 49 cytoplasmic aa (SR-A(Delta1-49)), which abolished SR-A-mediated ligand internalization without reducing cell adhesion. To identify additional cytoplasmic motifs within the first 49 aa that regulate SR-A function, the acidic residues in a conserved motif (EDAD) were changed to their amide derivatives (SR-A(QNAN)). The function and regulation of SR-A(QNAN) were compared with that of SR-A(Delta1-49) and SR-A in transfected HEK-293 cells. Blocking PI3K activation inhibited SR-A, but not SR-A(Delta1-49)- or SR-A(QNAN)-mediated cell adhesion. Although deleting (SR-A(Delta1-49)) or mutating (SR-A(QNAN)) the EDAD motif abolished the PI3K sensitivity of SR-A-mediated cell adhesion, these mutations did not affect ligand internalization or PI3K activation during cell adhesion. To define the mechanism by which PI3K regulates SR-A-mediated cell adhesion, the cellular localization of wild-type and mutant SR-A was examined. PI3K inhibition reduced surface localization of SR-A but not of SR-A(Delta1-49) or SR-A(QNAN). The regulation of SR-A surface localization by PI3K was confirmed in peritoneal macrophages, which endogenously express SR-A. Together, these results suggest a pathway in which SR-A binding to an immobilized ligand activates PI3K to recruit more receptor to the plasma membrane and enhances cell adhesion.