In the previous study (Sato K.-I. et al. (1997) FEBS Lett. 410, 136-140), we showed that the phosphorylation of Shc protein by c-Src is dependent on the binding of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) to the PTB domain of Shc. In this study, we demonstrate that, in contrast to c-Src, v-Src and epidermal growth factor (EGF) receptor can phosphorylate Shc in a PtdIns(4,5)P2-independent manner and at different phosphorylation sites. To determine the phosphorylation sites in Shc, we used mutant Shc proteins in which tyrosine residues (Y) 317 and/or 239 and 240 were replaced by phenylalanine residues (F). We found that Y317F Shc but not Y239/240F or Y239/240/317F Shc was phosphorylated by c-Src. The reaction was PtdIns(4,5)P2-dependent and inhibited by the addition of PTB domain of Shc. On the other hand, v-Src and EGF receptor were able to phosphorylate both Y317F and Y239/240F but not Y239/240/317F Shc in a PtdIns(4,5)P2-independent manner. These results highlight the difference between c-Src and v-Src or EGF receptor and suggest that c-Src can phosphorylate predominantly on Tyr239/240 of Shc only when Shc PTB domain is bound to PtdIns(4,5)P2.