Volatile benzenoids, including methyl p-methoxybenzoate, p-anisaldehyde, and p-anisalcohol, are responsible for the sweet and characteristic fragrance of loquat (Eriobotrya japonica, Rosaceae) flowers. Although the full pathway of volatile benzenoid synthesis has yet to be elucidated, their chemical structures suggest that O-methyltransferases are present in loquat and function in the methylation of the para-OH groups. In the present study, we used RNA-sequencing to identify four loquat genes (EjOMT1, EjOMT2, EjOMT3, and EjOMT4) that encode O-methyltransferases. We found that EjOMT1 was highly expressed in floral tissues, with an expression pattern that coincided with changes in intracellular volatile benzenoids during flower development. Recombinant EjOMT1 protein expressed in Escherichia coli showed the highest activity towards guaiacol with a K-m value of 35 mu M. Furthermore, the protein also showed lesser activities towards guaiacol-type benzenoids including eugenol, isoeugenol, vanillin, and ferulic acid, in addition to much weaker activities towards catechol and p-hydroxybenzenoid derivatives. However, no activity was shown towards phenylpropenes without m-methoxy substitution, t-anol and chavicol. Taken together, our findings indicate that EjOMT1 has a broad substrate specificity towards compounds with both para-OH and meta-OCH3 groups, unlike previously characterized O-methyltransferases for volatile benzenoid/phenylpropanoid biosynthesis. (C) 2016, The Society for Biotechnology, Japan. All rights reserved.