The presence of two short signature sequence motifs (His-Ile-Gly-His (HIGH) and Lys-Met-Ser-Lys (KMSK)) is a characteristic of the class I aminoacyl-tRNA synthetases. These motifs constitute a portion of the catalytic site in three dimensions and play an important role in catalysis. In particular, the second lysine of the KMSK motif (K2) is the crucial catalytic residue for stabilization of the transition state of the amino acid activation reaction (aminoacyl-adenylate formation). Arginyl-tRNA synthetase (ArgRS) is unique among all of the class I enyzmes, as the majority of ArgRS species lack canonical KMSK sequences. Thus, the mechanism by which this group of ArgRSs achieves the catalytic reaction is not well understood. Using three-dimensional modeling in combination with sequence analysis and site-directed mutagenesis, we found a conserved lysine in the KMSK-lacking ArgRSs upstream of the HIGH sequence motif, which is likely to be a functional counterpart of the canonical class I K2 lysine. The results suggest a plausible partition of the ArgRSs into two major groups, on the basis of the conservation of the HIGH lysine.