These two effects appear to be more important than the steric hindrance caused by the additional methoxy groups. Mut2 introduces two larger aromatic residues into the active site in close proximity to the phenolic or catecholic ring, and the catalytic H161. The quadruple mutant Mut4 is the most active towards 2c and 2d and also very active towards 2b. This variant stands out in its relatively high activity towards 1c and 1d. In contrast, the activity towards resveratrol is lower than that of the wild-type enzyme, and the activity towards pinostilbene is very low. Mut6 has the single substitution L117F, Mut1 the substitution F311W, and the double variant Mut2 combines both (L117F/F311W). While Mut6 has the highest activities towards resveratrol and shows good activity towards the phenolic acids 1c and 1d, it is neutral towards the phenolic styrenes. The triple variant Mut5 has an arginine in the hydrophobic active site. This variant has consistently low activity towards all substrates. Without experimentally determined structural information, accurate explanations of the effect of the amino acid substitutions in the hydrophobic active site cavity on the O-transmethylation activity are difficult. VvROMT is a dimer, where amino acids from one monomer contribute to the active site of the other. This constellation makes accurate predictions of substrate binding and -targeting exceedingly challenging. Our results highlight