Stevia rebaudiana leaves accumulate a mixture of at least eight different steviol glycosides. The pattern of glycosylation heavily influences the taste perception of these intensely sweet compounds. The majority of the glycosides are formed by four glucosylation reactions that start with steviol and end with rebaudioside A. The steps involve the addition of glucose to the C-13 hydroxyl of steviol, the transfer of glucose to the C-2' and C-3' of the 13-O-glucose and the addition of glucose to the hydroxyl of the C-4 carboxyl group. We used our collection of ESTs, an UDP-glucosyltransferase (UGT)-specific electronic probe and key word searches to identify candidate genes resident in our collection. Fifty-four expressed sequence tags (ESTs) belonging to 17 clusters were found using this procedure. We isolated full length cDNAs for 12 of the UGTs, cloned them into an expression vector, and produced recombinant enzymes in Escherichia coli. An in vitro glucosyltransferase activity enzyme assay was conducted using quercetin, kaempferol, steviol, steviolmonoside, steviolbioside, and stevioside as sugar acceptors, and (14)C-UDP-glucose as the donor. Thin layer chromatography was used to separate the products and three of the recombinant enzymes produced labelled products that co-migrated with known standards. HPLC and LC-ES/MS were then used to further define those reaction products. We determined that steviol UGTs behave in a regioselective manner and propose a modified pathway for the synthesis of rebaudioside A from steviol.