The determination of protein tyrosine phosphatase (PTP) activity using different protein substrates such as modified lysozyme or myelin basic protein is greatly affected by the type of enzyme involved, the condition of the assay, and the presence of effectors. The purpose of this study was to develop a general substrate of wide applicability with which variations in enzymatic activity would be minimized. A nonapeptide ENDYINASL derived from a highly conserved region of the T-cell phosphatase TC.PTP (Cool et al. (1989) Proc. Natl. Acad. Sci. USA 86, 5257-5261) was phosphorylated with a recombinant tyrosine kinase domain of the EGF receptor and purified on a (\mathrm{C} 18) cartridge. Phosphatase activities of intracellular and receptor-linked PTPs were as high as the highest values obtained with the protein substrates. The intracellular, low-molecularweight PTPs exhibited (K_{m}) values between 0.5 and 1.3 (\mu \mathrm{M}), whereas the receptor forms CD45 and RPTP (\alpha) gave values of 14 and (35 \mu \mathrm{M}), respectively. All PTPs displayed similar properties toward the peptide inciuding a low pH optimum and inhibition by vanadate, divalent cations, and heparin. Following immunoprecipitation, 1 ng of TC.PTP could be detected with ENDY(P)INASL compared to (10 \mathrm{ng}) in presence of protein substrates. (c) 1993 Academic Press, Inc.