Ternatins are blue anthocyanins found in the petals of Clitoria ternata (butterfly pea). Among them, ternatin C5 (delphinidin 3-O-(6''-O-malonyl)-beta-glucoside-3',5'-di-O-beta-glucoside; 2) has the structure common to all the ternatins, which is characterized by its glucosylation pattern: a 3,3',5'-triglucosylated anthocyanidin. In the course of studying biosynthetic pathways of ternatins, the key enzymatic activities to produce ternatin C5 were discovered in a crude enzyme preparation from the petals of a blue petal line of C. ternatea. When this preparation was tested for activity against several delphinidin glycosides, delphinidin 3-O-(6''-O-malonyl)-beta-glucoside-3'-O-beta-glucoside (6), a postulated intermediate, was found in the reaction mixture, together with three known anthocyanins, which were spectroscopically structurally identified. As a result of structural identification, the following enzymatic activities were identified: UDP-glucose :delphinidin 3-O-(6''-O-malonyl)-beta-glucoside-3'-O-beta-glucoside 5'-O-glucosyltransferase (5'GT), UDP-glucose :delphinidin 3-O-(6''-O-malonyl)-beta-glucoside 3'-O-glucosyltransferase (3'GT), UDP-glucose :delphinidin 3-O-glucosyltransferase, and malonyl-CoA :delphinidin 3-O-beta-glucoside 6''-malonyltransferase. In a mauve petal line, which did not accumulate ternatins but delphinidin 3-O-(6''-O-malonyl)-beta-glucoside in its petal, there were neither 5'GT nor 3'GT activities. Thus, the early biosynthetic pathway of ternatins may be characterized by the stepwise transfer of two glucose residues to 3'- and 5'-position of delphinidin 3-O-(6''-O-malonyl)-beta-glucoside (1; Scheme) from UDP-glucose.