Primordial aminoacyl-tRNA synthetases (aaRSs) based on the Rossman nucleotide binding fold of class I enzymes or the seven-stranded antiparallel beta-sheet fold of class II enzymes have been proposed to predate the contemporary aaRS. As part of an inquiry into class II aaRS evolution, the individual domains of the homodimeric Escherichia coli histidyl-tRNA synthetase (HisRS) were separately expressed and purified to determine their individual contributions to catalysis. A 320-residue fragment (Ncat HisRS) truncated immediately following motif 3 catalyzes both the specific aminoacylation of tRNA and pyrophosphate exchange, albeit less efficiently than the full-length enzyme. Ncat HisRS showed no mischarging of noncognate tRNAs but exhibited reduced selectivity for the C73 discriminator base, a principal aminoacylation determinant for histidine tRNAs. Size exclusion chromatography showed that Ncat HisRS is monomeric, indicating that the C-terminal domain is essential for maintaining the dimeric structure of the enzyme. The stably folded C-terminal domain (Cter HisRS) was inactive for both reactions and did not enhance the activity of Ncat HisRS when added in trans. The fusion of one or more accessory domains to a primordial catalytic domain may therefore have been a critical evolutionary step by which aminoacyl-tRNA synthetases acquired increased catalytic efficiency and substrate specificity.