Resistance of leukemia cells toL-asparaginase is presumed to be due to increased expression of asparagine synthetase activity by resistant cells, so they are no longer dependent on an exogenous source ofL-asparagine for growth. The mechanism by which cells acquire the ability for increased enzyme expression, however, has not been clearly defined. Evidence presented here indicates that genomic alterations in the form of translocations, gene amplification, or increased P-glycoprotein expression, do not account for the phenotypic transformation fromL-asparaginase sensitivity toL-asparaginase resistance. Instead, both sensitive and resistant L5178Y cells contain immunoreactive material detected by Western blotting with an antiserum prepared against bovine pancreatic asparagine synthetase. This suggests that the mechanism of resistance might involve modification of asparagine synthetase inL-asparaginase-resistant cells by an as-yet-unidentified mechanism or by inhibition of enzyme activity in theL-asparaginase-sensitive cells.