The enzymes that aminoacylate tRNAs have been studied extensively and can be organized into two distinct classes based on signature sequences and the position of aminoacylation. The class I enzymes have canonical HIGH and KMSKS sequences as part of a Rossman fold nucleotide‐binding site. The tryptophan‐specific enzymes have been placed in class I based on analysis of the cognate genes from Escherichia coli, B. stearothermophilus, B. taurus, and Homo sapiens. An unidentified open reading frame (ORF) on Saccharomyces cerevisiaechromosome XV, HRE342, has 46% identity with the bovine tryptophanyl‐tRNA synthetase and possesses the appropriate signature sequences. The predicted molecular weight of the putative HRE342 protein also closely matched the expected monomer size of the S. cerevisiaeenzyme. The HRE342 ORF plus about 250 bp of 5′ and 3′ flanking sequence was amplified by polymerase chain reaction, cloned into a 2 μ based vector, and transformed into a host strain, S. cerevisiaeJG369.3B. Nucleotide sequence analysis of the clone confirmed the presence of HRE342. Extracts from transformed yeast have a 30‐ to 100‐fold increase in specific activity of the tryptophanyl‐tRNA synthetase. An HRE342 locus in a diploid strain, PTY33XPTY44, was disrupted with a LEU2insert. Sporulation and tetrad analysis of the HRE342::LEU2strain demonstrated that HRE342 is an essential gene. We conclude that HRE342 is the S. cerevisiaegene encoding the cytoplasmic tryptophanyl‐tRNA synthetase, WRS1. A search of the SaccharomycesGenome Database using amino acid sequences from other eukaryotic aminoacyl‐tRNA synthetases suggests there is sufficient similarity to identify both class I and class II genes. © 1997 by John Wiley & Sons, Ltd.