Histidine-tRNA synthetase is one of the smallest bacterial aminoacyl-tRNA synthetases. It is less than one-half the size of the largest aminoacyl-tRNA synthetases. The entire nucleotide sequence of the Escherichia coli hisS locus was determined. The coding region is comprised of 424 codons, and the sequence was determined for 200 nucleotides on the 5'- and 3'-sides of the coding region. The translated nucleotide sequence was confirmed extensively by independent amino acid sequence information obtained by Edman degradations of purified peptides and by measurements of peptide masses by fast atom bombardment mass spectrometry. A significant sequence alignment of four bacterial aminoacyl-tRNA synthetases was reported recently (Webster, T., Tsai, H., Kula, M., Mackie, G., and Schimmel, P. (1984) Science 226, 1315-1317). Although the four enzymes vary considerably in length, this match occurs within the first 100 amino acids of each of the four enzymes and is in the segment believed to be part of the catalytic core. But no strong alignment could be found of the histidine sequence with these four tRNA synthetase sequences. This enzyme may be derived, therefore, from a different progenitor. Previous work suggested that three places in the hisS 5'-noncoding sequence could be promoter sites for RNA polymerase (Eisenbeis, S. J., and Parker, J. (1982) Gene 18, 107-114). We detected a 1400-nucleotide RNA species by RNA blot analysis with a hisS-specific probe. S1 nuclease mapping demonstrated a 5'-end to the RNA species occurs at -67 +/- 1, relative to the first nucleotide of the coding region. This position coincides with the predicted start site for transcription from one of the previously proposed promoter sites.