p-Carboxymethyl- l-phenylalanine (pCMF), a phosphotyrosine (pTyr) mimetic that is resistant to protein tyrosine phosphatase hydrolysis, was cotranslationally incorporated into proteins in Escherichia coli using an orthogonal amber suppressor tRNA/aminoacyl-tRNA synthetase (aaRS) pair. The pCMF-specific aaRS was identified from a large library of Methanococcus jannaschii tyrosyl-tRNA synthetase active-site mutants by a combination of positive and negative genetic selections. When pCMF was substituted for Tyr701 in human signal transducer and activator of transcription-1 (STAT1), a constitutively active mutant was obtained that dimerizes and binds a DNA oligonucleotide duplex that contains the M67 site recognized by Tyr701-phosphorylated STAT1. Genetic incorporation of pCMF into proteins should provide a new tool for the preparation of stable analogues of a wide array of phosphoproteins involved in signal transduction pathways, as well as the development of peptide-based, cellularly expressed inhibitors of pTyr binding proteins.