β-Glucosidase zymograms of certain maize genotypes (nulls) do not show any activity bands after electrophoresis. We have shown that a chimeric lectin called β-glucosidase aggregating factor (BGAF) is responsible for the absence of β-glucosidase activity bands on zymograms. BGAF specifically binds to maize β-glucosidase isozymes Glu1 and Glu2 and forms large, insoluble complexes. Furthermore, we have previously shown that the N-terminal (Glu50-Val145) and the C-terminal (Phe466-Ala512) regions contain residues that make up the BGAF binding site on maize Glu1. However, sequence comparison between sorghum β-glucosidases (dhurrinases, Dhr1 and Dhr2), to which BGAF does not bind, and maize β-glucosidases, and an examination of the 3-D structure of Glu1 suggested that the BGAF binding site on Glu1 is much smaller than predicted previously. To define more precisely the BGAF binding site, we constructed additional chimeric β-glucosidases. The results showed that a region spanning 11 amino acids (Ile72-Thr82) on Glu1 is essential and sufficient for BGAF binding, whereas the extreme N-terminal region Ser1-Thr29, together with C-terminal region Phe466-Ala512, affects the size of Glu1-BGAF complexes. To determine the importance of each region for binding, we determined the dissociation constants (Kd) of chimeric β-glucosidase-BGAF interactions. The results demonstrated that the extreme N-terminal and C-terminal regions are important but not essential for binding. To confirm the importance of Ile72-Thr82 on Glu1 for BGAF binding, we constructed chimeric Dhr2 (C-11, Dhr2 whose Val72-Glu82 region was replaced with the Ile72-Thr82 region of Glu1). C-11 binds to BGAF, indicating that the Ile72-Thr82 region is indeed a major interaction site on Glu1 involved in BGAF binding. We also constructed mutant β-glucosidases to identify and define the contribution of individual amino acids in the above three regions to BGAF binding. In the N-terminal region (Ile72-Thr82), critical region for BGAF binding, Glu1 mutants K81E and T82Y failed to bind BGAF in the gel-shift assay and their frontal affinity chromatography (FAC) profiles were essentially similar to that of sorghum β-glucosidase (dhurrinase 2, Dhr2), a non-binder, indicating that these two amino acids within Ile72-Thr82 region are essential for BGAF binding. In the extreme N-terminal (Ser1-Thr29) and C-terminal (Phe466-Ala512) regions, N481E [substitution of asparagine-481 with glutamic acid (as in Dhr)] showed lower affinity for BGAF, whereas none of the single amino acid substitutions in the Ser1-Thr29 region showed any effect on BGAF binding indicating that these regions play a minor role. To further confirm the importance of lysine-81 and threonine-82 for BGAF binding, we produced a number of Dhr2 mutants, and the results showed that all four unique amino acids (isoleucine-72, asparagine-75, lysine-81, and threonine-82) of Glu1 in the peptide span Ile72-Thr82 are required to impart BGAF binding ability to Dhr2. The sequence comparison among plant β-glucosidases supports the hypothesis that BGAF binding is specific to maize β-glucosidases because only maize β-glucosidases have threonine at position 82.