Efficiencies of the incorporation of various nonnatural amino acids carrying aromatic side groups into streptavidin were examined. The aromatic amino acids were linked to a mixed dinucleotide, pdCpA, and the resulting aminoacyl pdCpAs were coupled with tRNACCCG(-CA) to afford chemically aminoacylated tRNACCCG＇s. Mutant streptavidin mRNA containing a CGGG 4 base codon at the Tyr83 site was. prepared and added to an Escherichia coli in vitro translation system with the aminoacyl tRNACCCG. The expression of the full-length mutant streptavidins was confirmed by a Western blot analysis, and their biotin binding activity was examined by a dot blot analysis. The Western blot analysis indicated that the efficiencies of the incorporation were higher for aromatic groups with straight configurations than those with widely expanded or bend configurations. The incorporation efficiencies were also examined in a rabbit reticulocyte lysate. In the latter system, the efficiencies were markedly improved for nonnatural amino acids with large side groups such as pyrene and anthraquinone.