There is little known about effects to be expected from high intake of flavonoids with respect to regulation of glucose/glycogen homeostasis. Glucose/glycogen homeostasis is mainly regulated by glycogen synthase (GS) and glycogen phosphorylase (GP). We investigated effects of naturally occurring flavonoids with varying substitution pattern on the activity of isolated muscle GP. Almost all flavonoids tested inhibited phosphorylated, active GPa, as well as unphosphorylated, adenosine monophosphate-activated GPb. However, inhibition of GPa was two-to-fourfold stronger than that of GPb. The flavonol quercetin and the anthocyanidins cyanidin and delphinidin turned out to be the most potent inhibitors of GPa, with concentration values where enzymatic activity is 50% of the respective control in the low micromolar range (<5 microM). Furthermore, by comparing GPa inhibitory activity of typical representatives from all known flavonoid classes, structural elements of flavonoids required for effective GP inhibition could be identified.