Protoplasts from the mesophyll of barley primary leaves glycosilate and accumulate exogenous coumarin-aglycons such as scopoletin and esculetin. The corresponding glycosides are ultimately compartmentalized in the vacuoles. Glycosilation of the aglycons and accumulation of glycosides are markedly enhanced by light. The activity of UDP-glucose: esculetin glycosyltransferase is located in the cytosol. Isolated vacuoles take up the glucoside esculin. Transport across the tonoplast is dependent on ATP, is characterized by its saturation kinetics (K(m) = 0.22 mM), and is inhibited by the protonophore CCCP. A model for glycoside transport across the tonoplast and retention in the cell sap is proposed.