A tylosin-producer, Streptomyces fradiae, was transformed with plasmids carrying genes from Streptomyces thermotolerans that are involved in acyl modification of macrolide antibiotics. A transformant with pMAB3, in which macrolide 4"-O-acyltransferase gene (acyB1) and its regulatory gene (acyB2) are subcloned, produced several types of 4"-O-acyltylosins. A transformant with pAB11 delta EH containing macrolide 3-O-acyltransferase gene (acyA) in addition to the above two genes produced 3-O-acetyltylosin and 3-O-acetyl-4"-O-acyltylosins. Among the products of the latter transformant, 3-O-acetyl-4"-O-isovaleryltylosin (AIV) was detected as a minor component. When L-leucine, a precursor of isovaleryl-CoA, was added to the medium at the late stage of the fermentation, AIV content among the total macrolides increased ten-fold and AIV became a main product. This fact suggests that a high level of endogenous isovaleryl-CoA may be essential for the selective production of AIV by S. fradiae carrying pAB11 delta EH.