Although a pathway for betacyanin biosynthesis has been postulated, most of the catalytic steps have not yet been identified or demonstrated with biochemical evidence. In the postulated pathway, the glucose moiety of betanin is conjugated to the aglycone, betanidin, because the glucosyltransferase (GT) activity that produces betanin has been reported and its cDNA isolated. However, another pathway for betacyanin biosynthesis is proposed in which betanin is formed by GT acting at the 5,6-dihydroxyindoline-2-carboxylic acid (cyclo-DOPA) step, followed by condensation of the product with betalamic acid. Here, we show that GT activity acts upon cyclo-DOPA in the betacyanin synthetic pathway. A crude extract from the petals of four o'clocks (Mirabilis jalapa L.) was mixed with cyclo-DOPA and UDP-glucose. After the reaction was stopped with phosphoric acid, the product was chemically reacted with betalamic acid. In the final reaction mixture, betanin formation was confirmed by HPLC analysis, demonstrating cyclo-DOPA 5-O-glucosyltransferase activity. This activity was correlated with the accumulation of betanin during the development of four o'clock flowers and was detected in another five species of Centrospermae. These results indicate that the glucose moiety of betanin is introduced at the cyclo-DOPA step, which is followed by condensation with betalamic acid, and not at the betanidin aglycone step.