The glucosyl transfer reaction of kojibiose phosphorylase (KP; EC 2.4.1.230) was examined using glycerol or myo-inositol as an acceptor. In the case of glycerol, KP produced two main transfer products: saccharides A and B. The structure of saccharide A was O-alpha-D-glucopyranosyl-(1 -> 1)glycerol and that of saccharide B was O-alpha-D-glucopyranosyl-(1 -> 2)-O-alpha-D-glucopyranosyl-(1 -> 1)-glycerol. These results show that KP transferred a glucose residue to the hydroxyl group at position I of glycerol. On the other hand, when myo-inositol was used as an acceptor, KP produced four transfer products: saccharides 1-4. The structures of saccharides 1 and 2 were O-alpha-D-glucopyranosyl-(1 -> 1)- and O-alpha-D-glucopyranosyl-(1 -> 5)-myo-inositol, respectively; those of saccharides 3 and 4 were O-alpha-D-glucopyranosyl-(1 -> 2)-O-alpha-D-glucopyranosyl-(1 -> 1)-and O-alpha-D-glucopyranosyl-(1 -> 2)-O-alpha-D-glucopyranosyl-(1 -> 5)-myo-inositol, respectively. KP transferred a glucose residue to the hydroxyl group at position 1 or 5 of myo-inositol. On the basis of the structures of their glucosyl transfer products, glycerol and myo-inositol were found to have a common structure with three hydroxyl groups corresponding to the hydroxyl group of the glucose molecule at positions 2, 3 and 4. The conformation of these three hydroxyl groups in the structure is equatorial. This structure is the substrate recognition site of KP. It has been suggested that KP strictly recognizes the structures of glycerol and myo-inositol, and catalyzes the transfer reaction of a glucose residue to the hydroxyl group at position I in glycerol, and at position I or 5 in myo-inositol, corresponding to position 2 in glucose.