We have determined the sequence of cDNA for the human histidyl-tRNA synthetase (HRS) in a hepatoma cell line and confirmed it in fetal myoblast and fibroblast cell lines. The newly determined sequence differs in 48 places, including insertions and deletions, from a previously published sequence. By sequence specific probing and by direct sequencing, we have established that only the newly determined sequence is present in genomic DNA and we have sequenced 500 hundred bases upstream of the translation start site. The predicted amino acid sequence now clearly demonstrates all three motifs recognized in class 2 aminoacyl-tRNA synthetases. Alignment of E. coli, yeast, and when available, mammalian predicted amino acid sequences for three of the four members of the class 2a subgroup (his, pro, ser, and thr) shows strong preservation of amino acid specific signature regions proximal to motif 2 and proximal to motif 3. These probably represent the active site binding regions for the proximal acceptor stem and for the amino acid. The first two exons of human HRS contain a 32 amino acid helical motif, first described in human QRS, a class 1 synthetase, which is found also in a yeast RNA polymerase, a rabbit termination factor, and both bovine and human WRS, suggesting that it may be an RNA binding motif.