The structural gene for glutaminyl-tRNA synthetase has been isolated from a gene bank of Saccharomyces cerevisiae chromosomal DNA. Cloning was achieved by complementation of a recently described yeast strain that is auxotrophic for glutamine. A multicopy recombinant plasmid with a 5-kilobase-pair genomic insert conferred sixfold elevation in glutaminyl-tRNA synthetase activity and restored a Gln+ phenotype to strains that were Gln- by virtue of a mutant gln4 allele. Subfragments of the 5-kilobase insert directed integration of URA3 to GLN4. Further experiments established that GLN4 is an essential gene that is located on chromosome XV. RNA blots with a GLN4-specific probe detected a single transcript of approximately 2,900 nucleotides.