Aglycone flavonols are thought to modulate auxin transport in Arabidopsis thaliana via an as yet undefined mechanism. Biochemical studies suggest that flavonoids interact with regulatory proteins rather than directly with the PIN auxin efflux facilitator proteins. Auxin transport is enhanced in the absence of flavonoids (transparent testa4 [tt4]) and reduced in the presence of excess flavonols (tt7 and tt3). Steady state PIN mRNA levels in roots inversely correlate with auxin movement in tt mutants. PIN gene transcription and protein localization in flavonoid-deficient mutants appear to be modulated by developmental cues and are auxin responsive. Modulation of PIN gene expression and protein distribution by localized auxin accumulations occurs in the wild type as well. Flavonoids inhibit auxin transport primarily at the shoot apex and root tip and appear to modulate vesicular cycling of PIN1 at the root tip. In some auxin-accumulating tissues, flavonoid increases and changes in flavonoid speciation are subsequent to auxin accumulation.