A general biosynthetic method has been developed which makes it possible to site-specifically incorporate unnatural amino acids with novel properties into proteins. In this approach the codon encoding the amino acid of interest is replaced with the “blank” nonsense codon UAG by oligonucleotide-directed mutagenesis. A suppressor tRNA that recognizes this codon is generated by run-off transcription and then chemically aminoacylated with the desired unnatural amino acid. Addition of the mutagenized gene and the aminoacylated suppressor tRNA to an in vitro extract capable of supporting protein biosynthesis generates a mutant protein containing the unnatural amino acid at the specified position. This methodology has recently been used to study the stability, specificity, and catalytic properties of a number of proteins. In these studies amino acids and analogues possessing altered hydrogen-bonding, electronic, and steric properties and unique backbone conformations have all been site-specifically incorporated into proteins. In addition, photoactivatable amino acids, isotopically labeled amino acids, and amino acids bearing biophysical probes have been inserted site-specifically. This chemistry increases our ability to carry out detailed physical organic studies on this important class of macromolecules.