Biosynthesis of the sesquiterpene rotundone in Vespolina grapes during berry ripening was investigated over two consecutive seasons, revealing that the compound accumulates from veraison to harvest and reaches relatively high concentrations (up to 5.44 μg/kg). Rotundone levels up to 1.91 μg/kg were also found in clones of Gruener Veltliner, a white grape variety known to give 'peppery' wines. These concentrations are higher than those reported for Syrah grapes and are similar to the levels found in some plants. Rotundone was shown to accumulate almost exclusively in berry exocarp, suggesting that skin contact during winemaking could be used to modulate the peppery character of red wine. However, rotundone yield after the winemaking process was relatively low. Indeed, only 10% of the rotundone present in grapes was extracted during fermentation, and only 6% was recovered in bottled wine. The results presented in this work provide key knowledge for manipulation of the peppery character of wine in order to optimize the intensity of this characteristic wine aroma.