The isolation and characterization of a flavanone-specific 7-O-glucosyltransferase and its resolution from other glucosyltransferases in Citrus paradisi (grapefruit) seedlings is described. This new enzyme in the subclass 2.4.1 catalyzes the glucosylation of the 7-OH group of naringenin (4',5',7-trihydroxyflavanone) to prunin and has been purified (943-fold) by fractional precipitation with ammonium sulfate and successive chromatography on Sephadex G-100, hydroxyapatite, UDP-glucuronic acid agarose, Mono Q, and Mono P columns. It has a pH optimum of 7.5-8.0, an apparent pI of 4.3, and an apparent Mr of 54,900. This glucosyltransferase has an expressed specificity for the 7-position of the flavanones naringenin (Kmapp 62 microM; Kmapp UDPG 51 microM) and hesperetin (Kmapp 124 microM; Kmapp UDPG 243 microM) and did not accept other flavone or flavonol aglycones. Characteristics of other flavonoid glucosyltransferase activities found in grapefruit seedlings are also described.