The roles of O-methyltransferases (OMTs) in microorganisms are not well understood, and are suggested to increase antimicrobial activity. Studies on OMTs cloned from microorganisms may help elucidate their roles. Streptomyces coelicolor A3(2) produces many useful natural antibiotics such as actinorhodin. Based on sequence information from S. coelicolor A3(2) genome, it was possible to clone several methyltransferases. An OMT cloned from Streptomyces coelicolor A3(2), ScOMT1 was characterized by in vivo and in vitro assays. Of 23 compounds tested, 13 were found to serve as its substrates. Of the 13 substrates, the methylated positions of 7 compounds were determined by HPLC, NMR, and MS analyses. This OMT favored ortho-dihydroxyflavones. Among the compounds tested here, the best substrate is 6,7-dihydroxyflavone.