Flavonol 3-O-glucosides esterified with ferulic or p-coumaric acid at positions 3'' and 6'' are the major UV-B screening pigments of the epidermal layer of Scots pine (Pinus sylvestris) needles. The last steps in the biosynthesis of these compounds are catalyzed by enzymes that transfer the acyl part of hydroxycinnamic acid CoA esters to flavonol 3-O-glucosides. A newly developed enzyme assay revealed three flavonol 3-O-glucoside hydroxycinnamoyltransferases (HCTs) in Scots pine needles with specificities for positions 3'', 4'' or 6''. The positions of the acyl groups were identified by cochromatography with reference compounds and by NMR spectroscopy. The enzymes were characterized by molecular mass, isoelectric point, and also pH and temperature optima. Substrate specificities for flavonol glycosides and hydroxycinnamic acid CoA esters as well as kinetic properties of 3''- and 6''HCT suggested that acylation preferably occurs with glucosides and p-coumaroyl-CoA. In addition, acylation takes place in a well-defined order, beginning at position 6'' followed by acylation at position 3''. These results give the first detailed characterization of flavonol 3-O-glycoside HCTs involved in the protection of plant tissues against UV-B (280-315 nm) radiation.