An alternative to suppression of stop codons for the biosynthetic insertion of non-natural amino acids has been developed. Micrococcus luteus , a Gram-positive bacterium, is incapable of translating at least two codons. One of these unused codons was inserted in a gene to act as a nonsense site. An aminoacylated tRNA was synthesized which was complementary to this codon. The gene containing the missing codon was expressed in vitro in a M.luteus transcription/translation system. Read-through of the missing codon occurred only when the complementary tRNA was included. The results demonstrate that M.luteus can be used for incorporation of amino acids via synthetically prepared aminoacylated tRNAs. The use of a M. luteus translation system provides a method for incorporation of non-natural amino acids which avoids the use of stop codons.