The substrate specificity and the transglycosylation activity of neopullulanase was altered by site-directed mutagenesis on the basis of information from a threedimensional structure predicted by computer-aided molecular modeling. According to the predicted three-dimensional structure of the enzyme-substrate complex, it was most likely that Ile-358 affected the substrate preference of the enzyme. Replacing Ile-358 with Trp, which has a bulky side chain, reduced the acceptability of ␣-(136)-branched oligo-and polysaccharides as substrates. The characteristics of the I358W-mutated enzyme were quite different from those of wild-type neopullulanase and rather similar to those of typical starch-saccharifying ␣-amylase. In contrast, replacing Ile-358 with Val, which has a smaller side chain, increased the preference for ␣-(136)-branched oligosaccharides and pullulan as substrates. The transglycosylation activity of neopullulanase appeared to be controlled by manipulating the hydrophobicity around the attacking water molecule, which is most likely used to cleave the glucosidic linkage in the hydrolysis reaction. We predicted three residues, Tyr-377, Met-375, and Ser-422, which were located on the entrance path of the water molecule might be involved. The transglycosylation activity of neopullulanase was increased by replacing one of the three residues with more hydrophobic amino acid residues; Y377F, M375L, and S422V. In contrast, the transglycosylation activity of the enzyme was decreased by replacing Tyr-377 with hydrophilic amino acid residues, Asp or Ser.