Activation of the platelet-derived growth factor (PDGF) alpha receptor (alphaPDGFR) leads to cell migration and DNA synthesis. These events are preceded by the ligand-induced tyrosine phosphorylation of the receptor and its association with SH2-containing signaling enzymes including Src family members (Src), the phosphotyrosine phosphatase SHP-2, phosphatidylinositol 3-kinase (PI3K), and phospholipase C-gamma1 (PLCgamma). In this study, we sought to systematically evaluate the relative roles of the signaling enzymes that are recruited to the alphaPDGFR for DNA synthesis and cell migration. Our approach was to generate and characterize tyrosine to phenylalanine alphaPDGFR mutants that failed to associate with one or more of the above listed signaling enzymes. In a 3T3-like cell line (Ph cells), PDGF-dependent DNA synthesis was strictly dependent on only one of the receptor-associated proteins, PI3K. In contrast, multiple signaling enzymes were required for maximal chemotaxis, as receptors unable to associate with either Src, PI3K, or PLCgamma initiated chemotaxis to 4, 47, or 56% of the wild-type level, respectively. Furthermore, coexpression of mutant receptors revealed that these signaling enzymes do not need to be on the same receptor for a cell to respond chemotactically to PDGF. We conclude that for the alphaPDGFR, PI3K plays a major role in initiating DNA synthesis, whereas PI3K, PLCgamma, and especially Src are required for chemotaxis.