Phosphonomethyl phenylalanine (Pmp) is a non-hydrolyzable phosphotyrosyl (pTyr) mimetic, which has been incorporated into eleven-mer Pmp-containing peptides that have previously been reported to competitively inhibit the protein-tyrosine phosphatases PTP1 and PTP 1B. We have recently shown that phosphonodifluoromethyl phenylalanine (F2Pmp) is superior to Pmp as a pTyr mimetic in SH2 domain-binding peptides. Herein we find using the hexameric peptide sequence Ac-D-A-D-E-X-L-amide, where X = (D/L)-Pmp or L-F2Pmp, that the half maximal inhibition values of these two peptides against PTP 1B-mediated dephosphorylation of autophosphorylated insulin receptor to be 200 microM and 100 nM, respectively. These data indicate that F2Pmp induces a three orders of magnitude enhancement in affinity relative to Pmp, resulting in an exceptionally potent peptide-based PTP inhibitor. We conclude that F2Pmp may be a generally useful tool in the preparation of selective, high affinity PTP inhibitors.