Vanillate is converted to protocatechuate by an O -demethylase consisting of VanA and VanB in Streptomyces sp. NL15-2K. In this study, vanillate demethylase from this strain was functionally expressed in Escherichia coli , and its substrate range for vanillate analogs was determined by an in vivo assay using recombinant whole cells. Among aromatic methyl ethers, vanillate, syringate, m -anisate, and veratrate were good substrates, whereas ferulate, vanillin, and guaiacol were not recognized by Streptomyces vanillate demethylase. After vanillate, 4-hydroxy-3-methylbenzoate was a better substrate than m -anisate and veratrate, and the 3-methyl group was efficiently oxidized to a hydroxymethyl group. These observations suggest that the combination of a carboxyl group on the benzene ring and a hydroxyl group in the para -position relative to the carboxyl group may be preferable for substrate recognition by the enzyme. ¹H-NMR analysis showed that the demethylation product of veratrate was isovanillate rather than vanillate. Therefore, it was concluded that O -demethylation of veratrate by Streptomyces vanillate demethylase occurred only at the meta -position relative to the carboxyl group.