Two discrete furanocoumarin (5- and 8-)O-methyltransferases and a caffeic acid 3-O-methyl-transferase from cell cultures of Ruta graveoleus L. have been copurified by affinity chromatography on 1,6-diaminohexane agarose (AH-Sepharose 4B) linked with S-adenosyl-L-homocysteine (SAH). The furanocoumarin O-methyltransferases, which transfer a methyl group from S-adenosyl-L-methionine (SAM) to the 5- or 8-hydroxyls of linear furanocoumarins, were not retarded by 5-(3-carboxypropanamido)-xanthotoxin (CPAX) immobilized to AH-Sepharose 4B, but addition of SAM to the irrigant buffer led to complete retardation of both enzymes on this affinity system. An analogous phenomenon was observed for the caffeic acid O-methyltransferase, with a ferulic acid ligand coupled to the same insoluble support. SAH was as effective as SAM in promoting binding of the furanocoumarin O-methyltransferases to CPAX and caffeic acid 3-O-methyltransferase to immobilized ferulic acid, respectively. The strong and specific adsorption of these enzymes was abolished by exclusion of SAM or SAH from the irrigant buffer. It is concluded that the enzymes bind first to SAM or SAH, and that this binding process in turn induces the binding site for their specific phenolic substrates or their analogs. Based on these findings, a compulsory-ordered kinetic mechanism for the action of these O-methyltransferases is postulated.