Malto-oligosyltrehalose synthase (EC 5.4.99.15, MTSase) is a glycosyltransferase catalyzing the conversion of maltodextrins to malto-oligosyltrehaloses by forming α, α-1, l-glucosidic linkage. The enzyme slightly catalyzes the release of glucose from maltodextrins by hydrolysis of the α-1, 4 linkage of maltodextrins on the reducing end. The amino acid sequence of MTSase from Sulfolobus acidocaldarius ATCC 33909 showed significant homologies to those of not only other bacterial MTSases but also α-amylase family enzymes. The S. acidocaldarius MTSase lost the enzyme ac tivity by site-directed mutation of Asp228, G1u255, or Asp443 of the enzyme, which corresponded to the catalytic residues of α-amylase family enzymes. The mutation of Lys390 or Lys445 brought about a decrease of the transfer activity and an increase of the hydrolysis activity of the enzyme. The two lysine residues are conserved among MTSases but not α-amylase family enzymes. The tertialy structure analysis revealed that MTSase as well as α-amylase family enzymes contained a (β/α)₈ barrel structure as the catalytic domain. The three carboxyl residues (Asp228, G1u255 and Asp443) proposed as the catalytic center of MTSase were found to be located in the active cleft and positioned similarly to those of a -amylase family enzymes. There were two loops and one he lix structures on one end of the active cleft, where a nearly closed space was formed. The lysine residues (Lys390 and Lys445) were found to be situated in the closed structure. It was suggested that several residues containing the two lysines in the closed structure were involved in catalysis of the α, α-1, 1 transfer activity of MTSase.