Flaveria spp. accumulate flavonol sulfate esters whose biosynthesis is catalyzed by a number of position-specific flavonol sulfotransferases. Although the accumulation of sulfated flavonols appears to be tissue specific and developmentally regulated and to vary among related species, little is known about the mechanism of regulation controlling the synthesis of these metabolites. In the present work, we report the isolation of a cDNA clone from Flaveria bidentis (pBFST3) encoding flavonol 3-sulfotransferase (F3-ST), which catalyzes the first step in the biosynthesis of flavonol polysulfates. This clone (pBFST3) was expressed in Escherichia coli and produced an F3-ST with high affinity for the flavonol aglycones, quercetin, and its 7-methyl derivative, rhamnetin. In addition, the synthetic auxin 2,4-dichlorophenoxyacetic acid was shown to induce F3-ST enzyme activity and F3-ST mRNA transcript levels in cell cultures of F. bidentis. The F3-ST mRNA levels increased within the first 3 h, reaching a maximum after 24 h of treatment, and remained elevated for up to 48 h. Treatments with either quercetin 3-sulfate or quercetin 3,7,4'-trisulfate reduced F3-ST enzyme activity in cell cultures but had no effect on the transcript levels. These results are discussed in relation to the putative role of flavonoid conjugates in the regulation of auxin transport.