Wild-type petunia pollen accumulates high levels of flavonol 3-O-glycosides. Pollen from conditionally male-fertile petunia has no flavonols and is unable to germinate. Pollen function is restored both in vivo and in vitro by providing flavonol aglycones, but not flavonol glycosides, to the pollen. In the present study, incubation of an in vitro suspension of conditionally male-fertile pollen with kaempferol or quercetin resulted in the accumulation of kaempferol and quercetin 3-O-glycosides in the pollen. We identified two glycosyltransferase activities associated with the intact pollen grain that catalyze the formation of a gametophyte-specific class of flavonol glycosides. Feeding studies showed that product formation was highly specific for flavonols with an unsubstituted 3-hydroxyl group and was not dependent on an external source of UDP-hexose. Ultraviolet spectral analysis, fast atom bombardment mass spectrometry, 1H-nuclear magnetic resonance, and 13C-nuclear magnetic resonance identified the products as kaempferol and quercetin 3-O-(2"- O-beta-D-glucopyranosyl)-beta-D-galactopyranoside, identical with the flavonol 3-O-glycosides present in wild-type pollen. The sugars are linked in a 1-->2 configuration that results in a pollen-specific class of compounds. To retain both glycosyltransferase activities in a cell-free extract, it was necessary to add triton X-100, suggesting that one or both of the proteins may be associated with a pollen membrane. A model for flavonol glycoside biosynthesis and uptake into the pollen is discussed in terms of the germination requirement for flavonols.