Two identical cDNAs encoding glycyl-tRNA synthetase were isolated from K562 and HEL cell lambda gt11 libraries by screening with anti-EJ from a patient with dermatomyositis, previously shown to specifically inhibit this enzyme. The sequenced cDNA had 2,385 base pairs, with a predicted protein of 685 amino acids and M(r) 77,500. The antigen reactive with anti-EJ was immunoaffinity-purified from HeLa cells, and two proteolytic peptides matched areas of the predicted amino acid sequence. The sequence was 61.6% identical with that of silk moth glycyl-tRNA synthetase. Several very highly conserved regions were identified, including the single class II synthetase motif region and an N-terminal region similar to one found in at least three other synthetases. Near identity in other regions suggested that they also had important functions. Expression of the cDNA in Escherichia coli yielded a protein of expected size that reacted with the autoantibodies and was active as glycyl-tRNA synthetase.