Production of cyclo{-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->} (CTS, cyclic tetrasaccharide) from starch was attempted using 1,6-alpha-glucosyltransferase (6GT) and 1,3-a-isomaltosyltransferase (IMT) from Bacillus globisporus C11 The optimal conditions for production from partially hydrolyzed starch were as follows: substrate concentration, 3%; pH 67; temperature, 30degreesC; 6GT, 1 unit/g-dry solid (DS); IMT, 10 units/g-DS. The production of CTS was demonstrated and 544 g of CTS hydrate crystal powders were obtained from 3500 g of partially hydrolyzed starch. Two major by-products were also isolated from the reaction mixture and identified as the branched derivatives of CTSs, 4-O-alpha-D-glucopyranosyl-CTS and 3-O-alpha-isomaltosyl-CTS.