The phosphotyrosine binding (PTB) or phosphotyrosine interaction (PI) domain of the proto-oncoprotein p52SHC binds to an NPXpY consensus sequence found in several growth factor receptors (Kavanaugh, W. M., Turck, C. W., and Williams, L. T. (1994) Science 268, 1177-1179). The amino-terminal region of p52SHC, which includes the PTB/PI domain, has been previously shown to associate with protein-tyrosine phosphatase-PEST (PTP-PEST) in vivo (Habib, T. , Herrera, R., and Decker, S. J. (1994) J. Biol. Chem. 269, 25243-25246). We report here the detailed mapping of this interaction in a murine context using glutathione S-transferase fusion protein binding studies and peptide competition assays. We show that the interaction between murine SHC and murine PTP-PEST is mediated through the PTB/PI domain of murine SHC and an NPLH sequence found in the carboxyl terminus of murine PTP-PEST. Since this interaction is not dependent on the presence of a tyrosine-phosphorylated residue in the target sequence, this reveals that the PTB/PI domain of SHC can recognize both tyrosine-phosphorylated sequences and non-tyrosine-based recognition motifs.