Guard cells of the lower epidermis of leaflets of Vicia faba L. cv. Weißkernige Hangdown contain several kaempferol 3,7‐O‐glycosides. This was demonstrated for the first time by the use of isolated, highly purified guard cell protoplasts for flavonol estimation and quantitation. From a total of ca 12 kaempferol glycosides, three were identified by comparative thin layer chromatography and high performance liquid chromatography as kaempferol 3‐O‐glucoside 7‐O‐rhamnoside (major component), 3‐O‐rhamnogalactoside 7‐O‐rhamnoside and 3,7‐O‐bisglucoside (minor components). On average, the total flavonol content was estimated to be 85 fmol protoplast−1. From comparative investigations including alkaline‐induced (green) fluorescence characteristics of flavonols and UV‐microscopical studies we suggest that kaempferol glycosides are present in guard cells and epidermal cells in similar quantities, and that these compounds are in the vacuole.By contrast, mesophyll protoplasts have a low flavonol content (one sixth that of guard cells). In spite of the different total flavonol contents, individual components of each cell‐type are the same. However, they show differences in their quantitative distribution.