Regioselective 6-,7-,8-,3'-, and 4'-O-methylations underlie the structural diversity of lipophilic flavones produced in the trichomes of sweet basil (Ocimum basilicum L.). The positions 6, 7, and 4' are methylated by a recently described set of cation-independent enzymes. The roles of cation-dependent O-methyltransferases still require elucidation. Here, the basil trichome EST database was used to identify a Mg(2+)-dependent O-methyltransferase that was likely to accept flavonoids as substrates. The recombinant protein was found to be active with a wide range of o-diphenols, and methylated the 8-OH moiety of the flavone backbone with higher catalytic efficiency than the 3'-OH group of candidate substrates. To further investigate flavone 8-O-methylation, the activity of a putative cation-independent flavonoid 8-O-methyltransferase from the same EST collection was assessed with available substrate analogs. Notably, it was strongly inhibited by gardenin B, one of its expected products. The catalytic capacities of the two studied proteins suggest that two alternative routes to nevadensin, a major flavone in some basil cultivars, might exist. Correlating the expression of the underlying genes with the accumulation of 8-substituted flavones in four basil lines did not clarify which is the major operating pathway in vivo, yet the combined data suggested that the biochemical properties of flavone 7-O-demethylase could play a key role in determining the reaction order.