Four novel flavonol O-methyltransferases (OMTs) were partially purified from Chrysosplenium americanum by precipitation with ammonium sulfate, successive chromatography on Sephacryl S-200 and hydroxylapatite, and chromatofocusing on Polybuffer ion exchanger. They exhibited strict position specificity for positions 3 of quercetin, 7 of 3-methylquercetin, 4' of 3,7-dimethylquercetin, and 6 of 3,7,3'-trimethylquercetagetin. None of these enzymes reacted with phenylpropanoids, flavones, dihydroflavonols, or any of their glucosides. Except for the 7-OMT whose activity was lost during chromatofocusing, the other enzymes had apparent pI values of 4.8, 5.4, and 5.7 for the 3-, 4'-, and 6-OMTs, respectively. They had similar molecular weights (Mr 57,000) and their pH optima varied between 7.8 and 9.0, with a shift in optimal activity from lower to higher pH with increasing level of substrate methylation. Unlike the 3 and 4' enzymes, the 6-OMT showed an absolute requirement for Mg2+ whose activation was saturable and was inhibited by EDTA. The in vitro stepwise O-methylation of quercetin----3-methylquercetin----3,7-dimethylquercetin----3 ,7, 4'-trimethylquercetin tends to suggest a coordinated sequence of methyl transfers in this tissue.