Arabinogalactan proteins (AGPs) are plant proteoglycans that have been implicated in plant growth and development. The possible involvement of AGPs in the action of gibberellin (GA), a class of plant hormones, was examined by applying beta-glucosyl Yariv reagent (beta-Glc)3Y, a synthetic phenyl glycoside that interacts selectively with AGPs, to barley aleurone protoplasts. Gibberellin induces transcription and secretion of alpha-amylases in the protoplasts. Induction of alpha-amylase was clearly inhibited by (beta-Glc)3Y but not by (alpha-Gal)3Y, a negative control of the Yariv reagent that does not interact with AGPs. Transfection analysis, using an alpha-amylase promoter-GUS fusion gene in the protoplasts, indicated that the transcriptional activation of the alpha-amylase promoter was inhibited specifically by (beta-Glc)3Y. These observations are the first indication of an involvement of AGPs in a plant hormone function. The inhibitory effect of (beta-Glc)3Y was not observed when aleurone layers or half-seed grains were used. This result, together with the fact that protoplasts do not have cell walls, suggests that the AGPs that function in alpha-amylase induction reside at the plasma membrane. An aleurone-specific AGP was detected by reversed-phase HPLC, and supported the idea that an AGP may play an important role in aleurone-specific events. The possible mechanism of AGP function in gibberellin-induced alpha-amylase production is discussed.