Abstract Interspecific hybrids between Walker 256 carcinosarcoma rat cells which are asparagine requiring, and LMTK t mouse cells which are drug resistant and asparagine independent have been isolated. The hybrids were selectively isolated by taking advantage of the asparagine requirement, or, in some cases, combining the asparagine requirement with an azaguanine resistance marker. The hybrids: ( a) possessed a chromosome complement which was additive between the two parent lines; ( b) showed two marker chromosomes; ( c) possessed both rat and mouse forms of a number of different isozymes. The specific activities of asparagine synthetase was measured in the two parents and the hybrids. The enzyme level in the hybrids was found to be higher than the levels observed in the W 256 line, but only 10% of that observed in the LMTK −. The results are in agreement with, but do not prove, the hypothesis that asparagine requirement is due to a mutation in a structural cistron specifying the asparagine synthetase polypeptide.