The sesquiterpene biosynthesis in the exo- and mesocarp of grape berries (Vitis vinifera cultivar Lemberger) was investigated using in vivo feeding experiments with the key precursors [5,5-²H2]-mevalonic acid lactone (d2-MVL) and [5,5-²H2]-1-deoxy-D-xylulose (d2-DOX). The experiments demonstrate a de novo sesquiterpene biosynthesis in the exocarp, whereas no incorporation of the precursors was detectable in the mesocarp. Furthermore, sesquiterpenes were generated with a high incorporation rate via both pathways; the classical cytosolic mevalonic acid pathway as well as the plastidial DOXP/MEP route, demonstrating a substrate transport from plastid to cytoplasm.