An assay method that continuously measures the protein tyrosine phosphatase (PTP)-catalyzed dephosphorylation reaction based on fluorescence resonance energy transfer (FRET) was developed as an improvement of our previously reported discontinuous version [M. Nishikata, K. Suzuki, Y. Yoshimura, Y. Deyama, A. Matsumoto, Biochem. J. 343 (1999) 385-391]. The assay uses oligopeptide substrates that contain (7-metboxycoumarin-4-yl)acetyl (Mca) group as a fluorescence donor and 2,4-dinitrophenyl (DNP) group as a fluorescence acceptor, in addition to a phosphotyrosine residue located between these two groups. In the assay, a PTP solution is added to a buffer solution containing a FRET substrate and chymotrypsin. The PTP-catalyzed dephosphorylation of the substrate and subsequent chymotryptic cleavage of the dephosphorylated substrate results in a disruption of FRET, thereby increasing Mca fluorescence. In this study, we used FRET substrates that are much more susceptible to chymotryptic cleavage after dephosphorylation than the substrate used in our discontinuous assay, thus enabling the continuous assay without significant PTP inactivation by chymotrypsin. The rate of fluorescence increase strictly reflected the rate of dephosphorylation at appropriate chymotrypsin concentrations. Since the continuous assay allows the measurement of initial rate of dephosphorylation reaction, kinetic parameters for the dephospborylation reactions of FRET substrates by Yersinia, T-cell and LAR PTPs were determined. The continuous assay was compatible with the measurement of very low PTP activity in a crude enzyme preparation and was comparable in sensitivity to assays that use radiolabeled substrates. (c) 2006 Elsevier SAS. All rights reserved.