Prior methods for the chemical synthesis of phosphotyrosine-containing peptides involved the incorporation of fully protected phosphoamino acids into the peptide chain or phosphorylation of free phenol side chains after peptide assembly is complete. The present work describes a novel and general methodology for the solid-phase synthesis of phosphopeptides, featuring direct incorporation of Nα-(9-fluorenylmethyloxycarbonyl)-O-phospho-l-tyrosine (unprotected side chain). This technique obviated the formation of peptide byproducts containing tyrosine H-phosphonate, a previously unrecognized side reaction from literature phosphitylation/oxidation approaches. Phosphopeptides corresponding to the tyrosine phosphorylation site of adipocyte lipid binding protein were synthesized by the newer, preferred method. These peptides were purified and characterized by high-performance liquid chromatography (HPLC), capillary zone electrophoresis (CZE), amino acid analysis (AAA), fast atom bombardment mass spectrometry (FABMS), and 31P nuclear magnetic resonance (31P NMR). The synthetic peptides were tested as substrates for two distinct protein tyrosine phosphatases, rat brain protein tyrosine phosphatase (PTPase) and human acid phosphatase. Substrate specificity was measured at pH 6.0 and 37 °C, using a colorimetric assay for released inorganic phosphate. Kinetic analysis revealed that both the rat brain PTPase and the human adipocyte acid phosphatase catalyzed peptide dephosphorylation but with different rates and affinities. The rat brain PTPase displayed classical Michaelis-Menten kinetics, with Km's of 68 ± 9 μM and 42 ± 11 μM and kcat/Km values of 4.9 × 105 s-1 M-1 and 6.9 × 105 s-1 M-1 determined for phosphorylated peptides of lengths 4 and 10 residues, respectively. In contrast, the human acid phosphatase demonstrated linear kinetics, with no saturation observed up to 2 mM phosphopeptide. These results demonstrate the utility of the methodology and describe how synthetic tyrosine phosphopeptides can be used to assess the catalytic efficacies of enzymes that hydrolyze protein phosphotyrosine.