A unique, coleopteran-active protein, termed eCry3.1Ab, was generated following variable-region exchange of a Bacillus thuringiensis lepidopteran-active protein, Cry1Ab, with a Cry3A region. Our results support the hypothesis that this variable-region exchange is responsible for imparting strong bioactivity against the larvae of western corn rootworm (WCR) (Diabrotica virgifera virgifera LeConte), a pest species which is not susceptible to either parent protein sequence. This study demonstrates the potential of successfully engineering a portion(s) of a lepidopteranactive B. thuringiensis sequence so that it has activity against coleopterans. Further elucidation of the eCry3.1Ab activity indicated the importance of variable regions 4 to 6 that were derived from Cry1Ab instead of Cry1Ac. There was some flexibility in making domain III of engineered hybrid insecticidal proteins even more Cry1Ab-like and retaining activity, while there was less flexibility in making domain III more Cry3A-like and retaining activity. In vitro binding studies with brush border membrane vesicles demonstrated that there was specific binding of chymotrypsin-processed modified Cry3A (mCry3A), which was not diminished by addition of a 100-fold molar excess of chymotrypsin-processed eCry3.1Ab or unprocessed eCry3.1Ab. In addition, in the converse experiment, specific binding of chymotrypsin-processed eCry3.1Ab was not diminished by the presence of a 75-fold molar excess of chymotrypsin-processed mCry3A. These data support the hypothesis that eCry3.1Ab can interact with different binding sites than the activated form of mCry3A in the WCR brush border and may provide a different mode of action from the standpoint of resistance management.The three-dimensional structures of Bacillus thuringiensis ␦-endotoxins indicate that the Cry1, Cry2, Cry3, and Cry4 types of proteins have similar three-domain organizations (2, 10, 16, 18). The three structural domains are N-terminal domain I comprised of 7 ␣-helices, domain II containing three ␤-sheets in a so-called Greek key conformation, and C-terminal domain III, which is a ␤-sandwich in a so-called jellyroll conformation. The active portions of B. thuringiensis Cry proteins are also characterized by having five conserved blocks in their amino acid sequences, which are designated CB1 to CB5 from the N terminus to the C terminus (11). CB1 comprises approximately 29 amino acids, CB2 comprises approximately 67 amino acids, CB3 comprises approximately 48 amino acids, CB4 comprises approximately 10 amino acids, and CB5 comprises approximately 12 amino acids. The sequences before and after these five conserved blocks are highly variable and thus are designated the "variable regions," which are designated V1 toV6.