Genetic experiments have indicated that asparagine auxotrophs of Escherichias coli K-12 can be made asparagine prototrophs at either of two sites on the chromosome and that wild-type strains require both sites to be mutated to produce asparagine auxotrophy. The former asn locus is now called asnA, and the new gene is designated asnB. The asnB gene is located near gal.AsnA+ asnB and asnA asnB+ strains were constructed, and the asparagine synthetic reaction was characterized in extracts. These studies revealed that the asnA gene codes for the enzyme previously described (H. Cedar and J.H. Schwartz, J. Biol. Chem. 244: 4112-4121, 1969), whereas the asnB gene is involved in the production of an enzyme which differs from the one previously described in its specific activity in extracts, its stability at low and high temperatures, and its apparent ability to use either glutamine or ammonia as amide nitrogen donor. Physiological studies showed that either enzyme alone is sufficient to allow a maximal growth rate under conditions of asparagine limitation.