An efficient system was developed for the co-expression of a yeast tRNATyr/tyrosyl-tRNA synthetase (TyrRS) pair in Escherichia coli. Analysis of suppression patterns using several sets of E. coli and lambda phage mutants indicated that the expressed yeast suppressor tRNATyr was aminoacylated only with tyrosine by its cognate yeast TyrRS and not by E. coli TyrRS or other aminoacyl-tRNA synthetases. This extra tRNA/TyrRS pair is expected to be a key bridgehead for developing an in vivo system for the site-directed incorporation of unnatural amino acids into proteins.