In mammalian cells, the nine aminoacyl-tRNA synthetases (aaRS) specific for the amino acids (aa) Glu, Pro, Ile, Leu, Met, Gln, Lys, Arg and Asp are associated within a multienzyme complex. Arginyl-tRNA synthetase (ArgRS) is characterized by the occurrence of two structurally distinct forms of that enzyme: a complexed (approximately 74 kDa) and a free (approximately 60 kDa) form. The cDNA encoding the 74-kDa species of ArgRS from Chinese hamster ovary cells has been isolated and sequenced. The deduced aa sequence shows 38% identity to the homologous bacterial enzyme but displays an N-terminal polypeptide extension composed of 73 aa, which is absent in the free form of mammalian ArgRS. Two regions of this extension are predicted to be alpha-helical, leading to the clustering of Leu and Ile residues on one side of the helices. This suggests that the N-terminal domain is involved in the assembly of the 74-kDa species of ArgRS within the multisynthetase complex through hydrophobic interactions. By using the isolated cDNA, a Northern blot analysis showed a single mRNA species. Thus, there is a possibility that the free and complexed forms of ArgRS are encoded by the same gene.