<jats:p>FK‐506 is a macrolide antibiotic with immunosuppressant activity. Structurally, this compound contains three methylated hydroxyl groups at C13, C15 and C31. Previous biosynthetic studies using stable isotope‐feeding experiments have established methionine as the source of the methyl for these methylated hydroxyl groups. Based on this information and also the availability of the 31‐<jats:italic>O</jats:italic> ‐desmethylFK‐506, a metabolic precursor for the biosynthesis of FK‐506, a <jats:italic>S</jats:italic> ‐adenosyl‐<jats:sc>l</jats:sc>‐methionine‐dependent enzyme assay was developed and the enzyme 31‐<jats:italic>O</jats:italic> ‐desmethylFK‐506 <jats:italic>O</jats:italic>:methyl‐transferase was isolated from an extract of <jats:italic>Streptomyces sp.</jats:italic> MA 6858 and purified to near homogeneity. 31‐<jats:italic>O</jats:italic>‐DesmethylFK‐506 <jats:italic>O</jats:italic>:methyltransferase is a monomeric protein with an apparent molecular mass of 30000 Da and a pI of 4.4. The first 38 N‐terminal amino acids have been sequenced and are H<jats:sub>2</jats:sub>N‐SDVVETLRLPNGATVAHVNAGEAQFLYREIFTDRXYLRH. Functionally, this enzyme has a requirement for Mg<jats:sup>2+</jats:sup> with an optimum temperature of 34°C and a pH of 7.4 for full activity. Moreover, it catalyses the methylation of 31‐<jats:italic>O</jats:italic> ‐desmethylimmunomycin as efficiently as its own natural substrate, 31‐<jats:italic>O</jats:italic> ‐desmethylFK‐506. Additionally, FKMT catalyzes the C31 transmethylation reaction of 13,31‐O‐bis‐desmethyl‐, 15,31‐<jats:italic>O</jats:italic>‐bisdesmethyl‐, 13,15,31‐<jats:italic>O</jats:italic>‐trisdesmethyl‐ and 31‐<jats:italic>O</jats:italic>‐19,22‐cyclic‐hemiketalimmunomycins, which are all structural analogues of FK‐506. The reaction is, however, completely blocked if the vicinal hydroxyl which is present at the C‐32 position of the 31‐O‐desmethylFK‐506 structure is replaced with azide, phosphate or other substituents. Finally, evidence is presented indicating the close similarity of FKMT and DIMT, a 31‐<jats:italic>O</jats:italic> ‐desmethyl‐immunomycin:<jats:italic>O</jats:italic> methyltransferase, previously isolated from a cell‐free extract of <jats:italic>Streptomyces hygroscopicus</jats:italic> var <jats:italic>ascomyceticus</jats:italic>, an immunomycin (ascomycin/FK‐520) producer.