To evaluate and characterize stevioside biosynthetic pathway in Stevia rebaudiana Bertoni cv Houten, two enzyme fractions that catalyze glucosylation of steviol (ent-13-hydroxy kaur-16-en-19-oic acid) and steviol-glucosides (steviol-13-0-glucopyranoside, steviolbioside and stevioside), utilizing UDP-glucose as the glucose donor, were prepared from the soluble extracts of S. rebaudiana leaves. Enzyme fraction 1, passed through DEAE-Toyopearl equilibrated with 50 millimolar K-phosphate pH 7.5, catalyzed the glucosylation to steviol and 19-0-methylsteviol, but not to isosteviol and 13-0-methylsteviol, indicating that 13-hydroxyl group of the steviol skeleton is glucosylated first from UDP-glucose to produce steviol-13-0-glucopyranoside. Enzyme fraction 11, eluted from the DEAE-Toyopearl column with 0.15 molar KCI, catalyzed the glucose transfer from UDP-glucose to steviol-13-O-glucopyranoside, steviolbioside and stevioside, but not to rubusoside (13,19-di-O-glucopyranoside) and rebaudioside A. The reaction products glucosylated from steviol-13-0-glucopyranoside, steviolbioside and stevioside were identified to be steviolbioside, stevioside and rebaudioside A, respectively. These results indicate that in the steviol-glucoside biosynthetic pathway, steviol-13-0-glucopyranoside produced from the steviol glucosylation is successively glucosylated to steviolbioside, then to stevioside producing rebaudioside A.One remarkable, sweet constituent of Stevia rebaudiana Bertoni, a wild shrub belonging to Compositae family, has been named stevioside, and proven to some 300 times as sweet as sucrose. Stevioside is a glucoside composed of three glucose molecules and an aglycon, steviol (ent-13-hydroxy kaur-16-en-19-oic acid), a diterpenoidic carboxylic alcohol (Fig. 1). In addition to stevioside, the leaves of S. rebaudiana contain several kinds of the steviol-related glucosides such as steviolbioside, rebaudioside A, and others as the minor constituents. Stevioside and rebaudioside A, the latter is stevioltetraglucoside, are known to be the main sweet constituents (10). As a consequence of their high content (5-10% of dry weight) in sweet glucosides, S. rebaudiana leaves are now becoming important in Japan, Brazil and other countries as a new noncaloric sweetner.Several works on the relationship between sweetness and chemical structures of steviol-glucosides (2,3,5,9,11,13) and the methods for chemical synthesis of stevioside (15)