The human gene for asparagine synthetase has been isolated and the molecular organization has been determined by mapping and DNA sequencing of intron-exon boundaries. The gene spans 35 kb and contains 13 exons. The structure of the human gene has a high degree of similarity to that of the hamster asparagine synthetase gene, with identical positions for all but one of the intron regions. The 5' upstream region of this gene, like other housekeeping genes, lacks conventional TATA and CAAT boxes. Comparisons of the 5' upstream sequences of the human and hamster genes show limited similarity; however, both have a very high G + C content which may play a role in expression through DNA methylation.