The gene for the yeast Saccharomyces cerevisiae glutamine tRNA synthetase is shown here to encode a protein of 809 amino acids. This contrasts with the 551 amino acids of the Escherichia coli glutamine tRNA synthetase. The yeast GLN4 transcripts have 5' termini that start approximately 25 nucleotides in front of the long open reading frame. Much of the extra size of the yeast enzyme is due to a large amino-terminal extension. At codon 225, the yeast enzyme aligns with the amino terminus of the E. coli protein. From this point on, the two sequences have an average of 40% identity, with a few small gaps for alignment, until their respective carboxyl termini. At codon 254 of the yeast and codon 30 of the E. coli enzyme, however, there starts an exact 15-amino acid match between the two proteins. This match encompasses and is partially the same as a short sequence which is a signature sequence for the amino acid group of the bacterial aminoacyl-tRNA synthetases which are specific for different amino acids. This is the strongest sequence match found between any yeast cytoplasmic or mitochondrial aminoacyl-tRNA synthetase with its bacterial homologue. This region of the structure is associated with a nucleotide fold. The result provides strong validation of the signature sequence, especially for sequences where the homology relationships are less dramatic than in this example. Because the 224-amino acid extension of the yeast enzyme does not align with any part of the E. coli enzyme, we propose that it is not associated directly with the catalytic function of the enzyme. Its possible function is investigated in the accompanying paper.