Cry1Ai-h-loop 2 is a mutant of Cry1Ai constructed by exchanging loop 2 from Cry1Ah protein and shows insecticidal activity against Helicoverpa armigera.  The toxicity of Cry1Ai-h-loop 2, in contrast to the very low toxicity of Cry1Ai, is closely associated with the eleven residues in the loop 2 region.  To characterize the key sites of loop 2 in Cry1Ai-h-loop 2, alanine-substituted mutants were generated.  The toxicity of these mutants against H. armigera indicated that dual-mutant on Gly³⁷³ and Asn³⁷⁵ caused a significant decrease in toxic activity.  ELISA binding and competition binding assays demonstrated that the reduction of toxicity in the mutant of interest was correlated with decreased binding affinity.