Three-dimensional structures of cyclodextrin glucanotransferases (CGTases) have revealed that four aromatic residues, which are highly conserved among CGTases but not found in alpha-amylases, are located in the active center. To analyze the roles of these aromatic residues, Phe-183, Tyr-195, Phe-259, and Phe-283 of Bacillus sp. 1011 CGTase were replaced by site-directed mutagenesis, and the effects of this procedure were examined. Y195L-CGTase, in which Tyr-195 was replaced by a leucine residue, underwent a drastic change in its cyclization characteristics: it produced considerably more gamma-cyclodextrin than the wild-type enzyme and virtually no alpha-cyclodextrin. Y195L-CGTase had increased Km values for cyclodextrins, whereas the values for a linear maltooligosaccharide donor were insignificantly changed. Taken together with the structural information of CGTase crystals soaked with substrates, we propose that Tyr-195 plays an important role in the spiral binding of substrate. Replacing either Phe-183 or Phe-259 with leucine induced increased Km values for acceptors. Furthermore, the double mutant F183L/F259L-CGTase had considerably decreased cyclization efficiency, but the intermolecular transglycosylation activity remained normal. These results indicated that Phe-183 and Phe-259 are cooperatively involved in acceptor binding, and that they play a critical role in cyclization when the nonreducing end of amylose binds to the active center of CGTase. Replacing Phe-283 with a leucine residue induced a decrease in kcat and in affinity for acarbose, suggesting that Phe-283 is involved in transition-state stabilization.