Two cDNA clones (SAS1 and SAS2) encoding different isoforms of asparagine synthetase (AS; EC 188.8.131.52) were isolated. Their DNA sequences were determined and compared. The amino-terminal residues of the predicted SAS1 and SAS2 proteins were identical to those of the glutamine binding domain of AS from pea, asparagus, Arabidopsis and human, suggesting that SAS1 and SAS2 cDNAs encode the glutamine-dependent form of AS. The open reading frames of SAS1 and SAS2 encode a protein of 579 and 581 amino acids with predicted molecular weights of 65182 and 65608 Da respectively. Similarity of the deduced amino acid sequences of SAS1 and SAS2 with other known AS sequences were 92% and 93% for pea AS1; 91% and 96% for pea AS2; 88% and 91% for asparagus; 88% and 90.5% for Arabidopsis; 70.5% and 72.5% for E. coli asnB and 61% and 63% for man. A plasmid, pSAS2E, was constructed to express the soybean AS protein in Escherichia coli. Complementation experiments revealed that the soybean AS protein was functional in E. coli. Southern blot analysis indicated that the soybean AS is part of a small gene family. AS transcript was expressed in all tissues examined, but higher levels were seen in stem and root of light-grown tissue and leaves of dark-treated tissue.