In the plant order Caryophyllales, the chromogenic anthocyanins have been replaced by functionally equivalent but biosynthetically different betacyanins (Clement & Mabry, 1996). The starting point of these investigations was the observation that up to now, only for one member of the Caryophyllales, Dorotheanthus bellidiformis (Livingstone daisy, Aizoaceae), two regiospecific betanidin glucosyltransferases have been characterized (Heuer et al., 1992, 1996; Vogt et al., 1997). As the accumulation of considerable amounts of cyclo-Dopa-5-O-glucoside had been reported for Beta vulgaris L. (Chenopodiaceae) by Wyler and co-workers in 1984, the question arose if glucosylation at the betanidin level is an exception or the rule in betacyanin biosynthesis. After development of an sensitive enzyme activity test, several plants from different families of the order Caryophyllales have been screened for activity of a putative cyclo-Dopa-glucosyltransferase, but all attempts to show the existence of such an enzyme activity failed. Furthermore investigation of cyclo-Dopa glucoside accumulation in B. vulgaris plants over eight weeks contradicted the previous published data by Wyler and co-workers (1984, 2001). Only trace amounts of this compound were detectable, most likely liberated from betanin that is in equilibrium with its hydrolytic products, cyclo-Dopa glucoside and betalamic acid (W. Schliemann, unpublished results). A molecular approach, based on polymerase-chain reaction and degenerated primers derived from the Plant Secondary Product GT consensus sequence, led to the first cloning and functional expression of two full-length cDNAs from B. vulgaris cell suspension culture with high homology to the corresponding enzymes from D. bellidiformis. The open reading frame (orf) of UGT73A4 encodes a protein of 476 amino acids with a molecular mass of 54 kDa, whereas the orf of UGT71F1 encodes a polypeptide of 492 amino acids with a comparable molecular weight, catalyzing the position-specific transfer of glucose from UDP-glucose to different flavonoids and to a very low extent to the chromogenic betanidin. In vitro studies on substrate specificity indicated that both enzymes exhibit a broad substrate spectrum regarding the sugar acceptor, preferring flavonoids with ortho-dihydroxy groups in the B-ring. In contrast the specificity for the sugar-donor was very strict, only UDP-glucose was accepted. From our studies it may be evident that glycosylation of betacyanins is a monophyletic phenomenon originating from enzymes previously involved in flavonoid metabolism. Expression analysis revealed that the UGT73A4 transcript accumulated not only in the cell culture of B. vulgaris, but also in young plants and the ancient form Beta maritia. The UGT71F1 mRNA could only be detected in the B. vulgaris cell suspension culture. Based on site-directed mutagenesis and a homology modelling approach, two 3D-models of the UGT73A5 from D. bellidiformis and the UGT73A4 from B. vulgaris have been developed. They provide a preliminary insight into the possible tertiary structure of two plant glucosyltransferases, the putative active site and are suitable to explain observed differences between the two homologous enzymes. Semiempirical calculations suggest a SN1-like mechanism of sugar transfer under inversion of configuration (α→β) for the UGT73A5 from D. bellidiformis, which may be seen as an alternative towards the SN2-mechanism proposed in literature (Kapitonov & Yu, 1999). Nevertheless the 3D-model structures presented here cannot substitute further investigations by spectroscopic methods, e.g. NMR, and their qualitiy may be finally judged after the first crystal structure of a plant glucosyltransferase is resolved. Further studies on the glucosyltransferase pool of different members of the Caryophyllales will be required to answer the question wether glucosylation at the betanidin level is the rule or an exception throughout betacyanin-synthesizing plants.