Until 30 years ago, it had been considered that D-amino acids were excluded from living systems except for D-amino acids in the cell wall of microorganisms. However, D-amino acids, in the form of free amino acids, peptides and proteins, were recently detected in various living organisms from bacteria to mammals. The extensive distribution of bio-functional D-amino acids challenges the current concept of protein synthesis: more attention should be paid to the stereospecificity of the translation machine. Besides aminoacyl-tRNA synthetases, elongation factor Tu and some other mechanisms, D-Tyr-tRNA(Tyr) deacylases provide a novel checkpoint since they specifically recycle misaminoacylated D-Tyr-tRNA(Tyr) and some other D-aminoacyl-tRNAs. Their unique structure represents a new class of tRNA-dependent hydrolase. These unexpected findings have far-reaching implications for our understanding of protein synthesis and its origin.