In an attempt to generate mutant aminoacyl-tRNA synthetases capable of charging noncanonical amino acids, a series of yeast tyrosyl-tRNA synthetase (TyrRS) mutants was constructed by site-specific mutagenesis of putative active site residues, which were deduced by analogy with those of Bacillus stearotherinophilus TyrRS. Among these mutants, one with the replacement of tyrosine at position 43 by glycine, “Y43G, ” was found to be able to utilize several 3-substituted tyrosine analogues as substrates for aminoacylation. The catalytic efficiency (k_cat/K_m) of mutant Y43G for aminoacylation with L-tyrosine was about 400-fold decreased as compared to that of the wild-type TyrRS. On the other hand, the ability to utilize 3-iodo-L-tyrosine was newly generated in this mutant TyrRS, since the wild-type TyrRS could not accept 3-iodo-L-tyrosine at all under physiological conditions. This mutant TyrRS should serve as a new tool for site-specific incorporation of non-canonical amino acids, such as those in 3-substituted tyrosine analogues, into proteins in an appropriate translation system in vivo or in vitro.