A strategy, termed alanine-scanning mutagenesis, was used to identify the amino acid residues which are critical to the antigenicity of Escherichia coli L-asparaginase (L-ASP). Three continuous alkaline residues, 195RKH197, were mutated to Ala selectively. Four mutant recombinant L-ASPs were constructed and expressed in E. coli, and then purified. The purified mutants showed a single band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were more than 95% pure by reverse high-performance liquid chromatography. The activities of wild-type and mL-ASPs in the fermentative medium were all about 130 U/mL. The change from 195RKH197 to 195AAA197 reduced the antigenicity of the enzyme greatly as shown in competition enzyme-linked immunosorbent assay using polyclonal antibodies raised against the wild-type L-ASP from rabbits. The results show that residues 195RKH197 of E. coli L-ASP are critical to its antigenicity.