We have constructed synthetic genes encoding different Escherichia coli suppressor tRNAs for use in amino acid substitution studies and protein engineering. We used oligonucleotides to assemble the genes for different tRNAs with the anticodon 5' CTA 3'. The suppressor genes are expressed from a synthetic promoter derived from the promoter sequence of the E. coli lipoprotein gene. The genes have been used to suppress an amber mutation in a protein coding sequence, and the resulting altered protein has been subjected to sequence analysis to determine the nature of the amino acid inserted at the amber site. Twelve amino acids can now be added in response to the amber codon. We have employed these suppressors to study amino acid substitutions in the lac repressor.