The 1-N-naphthylphthalamic acid (NPA)-binding protein is a putative negative regulator of polar auxin transport that has been shown to block auxin efflux from both whole plant tissues and microsomal membrane vesicles. We previously showed that NPA is hydrolyzed by plasma-membrane amidohydrolases that co-localize with tyrosine, proline, and tryptophan-specific aminopeptidases (APs) in the cotyledonary node, hypocotyl-root transition zone and root distal elongation zone of Arabidopsis thaliana (L.) Heynh. seedlings. Moreover, amino acyl-beta-naphthylamide (aa-NA) conjugates resembling NPA in structure have NPA-like inhibitory activity on growth, suggesting a possible role of APs in NPA action. Here we report that the same aa-NA conjugates and the AP inhibitor bestatin also block auxin efflux from seedling tissue. Bestatin and, to a lesser extent, some aa-NA conjugates were more effective inhibitors of low-affinity specific [3H]NPA-binding than were the flavonoids quercetin and kaempferol but had no effect on high-affinity binding. Since the APs are inhibited by flavonoids, we compared the localization of endogenous flavonoids and APs in seedling tissue. A correlation between AP and flavonoid localization was found in 5- to 6-d-old seedlings. Evidence that these flavonoids regulate auxin accumulation in vivo was obtained using the flavonoid-deficient mutant, tt4. In whole-seedling [14C]indole-3-acetic acid transport studies, the pattern of auxin distribution in the tt4 mutant was shown to be altered. The defect appeared to be in auxin accumulation, as a considerable amount of auxin escaped from the roots. Treatment of the tt4 mutant with the missing intermediate naringenin restored normal auxin distribution and accumulation by the root. These results implicate APs and endogenous flavonoids in the regulation of auxin efflux.