Information gains from the seed of the model plant Arabidopsis thaliana (Brassicaceae) have greatly contributed to a better understanding of flavonoid synthesis and may be used for crop improvement. However, exhaustive identification of the flavonoid accumulated in Arabidopsis seed was still lacking. Complementary investigations of seed flavonoids by LC-ESI-MS, LC-ESI-MS-MS, and NMR spectroscopy in Arabidopsis led to full characterization of monoglycosides, namely, quercetin 3-O-alpha-rhamnopyranoside and kaempferol 3-O-alpha-rhamnopyranoside, and diglycosides, namely, quercetin and kaempferol 3-O-beta-glucopyranoside-7-O-alpha-rhamnopyranoside and quercetin and kaempferol 3,7-di-O-alpha-rhamnopyranoside. Interestingly, the tt7 mutant that lacks flavonoid-3'-hydroxylase and consequently accumulates only kaempferol derivatives was shown to contain three additional products, kaempferol 3-O-beta-glucopyranoside, kaempferol 3-O-alpha-rhamnopyranoside-7-O-beta-glucopyranoside, and the triglycoside kaempferol 3-O-beta-[alpha-rhamnopyranosyl(1-->2)-glucopyranoside]-7-O-alpha-rhamnopyranoside. Taken together these results allow a scheme for flavonol glycosylation to be proposed.