UV-B radiation and water deficit may trigger flavonol and anthocyanin biosynthesis in plant tissues. In addition, previous research has showed strong qualitative effects on grape berry skin flavonol and anthocyanin profiles in response to UV-B and water deficit. The aim of this study is to identify the mechanisms leading to quantitative and qualitative changes in flavonol and anthocyanin profiles, in response to separate and combined UV-B and water deficit. Grapevines (Vitis vinifera L. cv. Tempranillo) were exposed to three levels of UV-B radiation (0, 5.98 and 9.66 kJ m⁻² day⁻¹) and subjected to two water regimes. A strong effect of UV-B on flavonol and anthocyanin biosynthesis was found, resulting in an increased anthocyanin concentration and a change in their profile. Concomitantly, two key biosynthetic genes (FLS1 and UFGT) were up-regulated by UV-B, leading to increased flavonol and anthocyanin skin concentration. Changes in flavonol and anthocyanin composition were explained to a large extend by transcript levels of F3′H, F3′5′H and OMT2. A significant interaction between UV-B and water deficit was found in the relative abundance of 3′4′ and 3′4′5′ substituted flavonols, but not in their anthocyanin homologues. The ratio between 3′4′5′ and 3′4′ substituted flavonols was linearly related to the ratios of F3′5′H and FLS1 transcription, two steps up-regulated independently by water deficit and UV-B radiation, respectively. Our results indicate that changes in flavonol profiles in response to environmental conditions are not only a consequence of changes in the expression of flavonoid hydroxylases; but also the result of the competition of FLS, F3′5′H and F3′H enzymes for the same flavonol substrates.