Abstract The phosphonodifluoromethyl phenylalanine (F 2Pmp) is superior to phosphonomethyl phenylalanine (Pmp) as a non-hydrolyzable phosphotyrosine (pTyr) mimetic. The difluoromethyl moiety increases the inhibitory potency of a F 2Pmp-containing peptide over a Pmp-containing counterpart by 1000-fold toward the protein tyrosine phosphatase (PTPase), PTP1. Fluorine substitution at the methylene carbon have the double effect of lowering the phosphonate pK a2 as well as introducing hydrogen bonding interactions similar to the phosphate ester oxygen in pTyr. The inhibition of PTP1-catalyzed dephosphorylation reaction by both the F 2Pmp and Pmp-containing peptides did not vary as a function of pH. The data indicate that both the monoanion and the dianion forms of the phosphonate bind PTP1 with equal efficiency. Thus, the better binding by the F 2Pmp-peptide as compared to the Pmp-peptide is not due to the difference in pK a2. Taken together, these results offer an explanation for the increased affinity of F 2Pmp for PTP1. The two fluorine atoms in F 2Pmp may be able to interact with active site residues in PTP1 in a fashion analogous to that involving the phenolic oxygen and side chains in the active site of PTP1. K i measurements for a simple phosphonic acid, Pmp- and F 2Pmp-containing peptides suggest that although the principal recognition element is F 2Pmp itself, the surrounding amino acids are required for high affinity binding. Comparative analysis of the inhibition of PTP1, PTPα and LAR by F 2Pmp-containing peptides suggests that selective, tight-binding PTPase inhibitors can be developed.