Hetero-branched cyclodextrins (CDs) were synthesized by transglycosylation or the reverse action of several enzymes such as βββ-galactosidases, α-galactosidases, α-mannosidases, lysozyme and R-Nacetylhexosaminidase. Their structures were analyzed by methylation, FAB-MS, and NMR spectroscopies. β-Galactosidase and a-galactosidase from microorganisms synthesized hetero-branched CDs, of which the galactose residues were linked at side chains of the branched CDs. But these enzymes could not synthesize galactosyl-CDs, directly linked to the CD ring. However, α-galactosidase from coffee bean and α-mannosidase and N-acetylhexosaminidase from jack bean could bind galactosyl, mannosyl and N-acetylglucosaminyl residues directly to the CD rings, respectively, by transglycosylation or reverse action. The effects of the side-chain residues in branched CDs, on solubility, hemolytic activity, and inclusion reactions with 6-O-α-D-glucosyl-CDs, 6-O-α-D-galactosyl-CDs, and 6-O-α-Dmannosyl-CDs, were examined.