We have shown previously that transcripts corresponding to the cDNA clone pBH72-F1, with similarities to O-methyltransferases (OMT), accumulated in barley leaves in response to attack by the pathogenic fungus Blumeria graminis (Plant Mol Biol 26 (1994) 1797). To investigate the accumulation pattern in the defence response and the organ localization of the pBH72-F1-encoded polypeptide (F1-OMT), an antiserum was raised against Escherichia coli expressed F1-OMT. The 43 kDa protein was absent in normal leaves but accumulated strongly in response to pathogen attack. The F1-OMT protein accumulated faster in barely lines inoculated with an avirulent B. graminis isolates compared to a virulent isolate. Additionally, F1-OMT related proteins were detected in developing kernels. F1-OMT was expressed as a functional enzyme in E. coli and the substrate specificity was investigated. The enzyme exhibited OMT activity towards flavonoid aglycones with the highest activity against apigenin (4',5,7-trihydroxyflavone). In contrast, caffeic acid did not serve a substrate for F1-OMT. The product of F1-OMT was analyzed by HPLC and GC-MS and found to be genkwanin (4',5-dihydroxy-7-methoxyflavone). Initial velocity data were best represented by a sequential bi-bi mechanism, and kinetic parameters of K(SAM) = 10.9 μM, K(apigenin) = 4.6 μM and a specific activity of 0.45 μkat/g were obtained. Barley F1-OMT, apigenin 7-O-methyltransferase, is suggested to be involved in the production of a methylated flavonoid phytoalexin.