The production of tylosin by Streptomyces fradiae KA-427 in a defined medium was inhibited by ammonium ions and by inorganic phosphate. The production of protylonolide, an early lactonic intermediate of tylosin biosynthesis with the same carbon skeleton as tylosin aglycone, by a mutant of strain KA-427 was also reduced by these two kinds of ions. In contrast, the bioconversion of protylonolide to tylosin by another mutant was less susceptible to ammonium ions but was sensitive to inorganic phosphate. The addition of protylonolide to a culture of S. fradiae KA-427 increased the tylosin yield, suggesting that aglycone synthesis is limiting under the conditions used. When L-valine, L-leucine, L-isoleucine, L-threonine, or the corresponding 2-keto acid was added to the culture medium, the protylonolide titer increased. The addition of [¹⁴C]valine gave rise to [¹⁴C]protylonolide. ¹³C NMR spectroscopic analysis revealed that iso-butyrate, which is a valine metabolite, was incorporated into protylonolide at the carbons known to originate from propionate and n-butyrate. Taking account of these findings, the regulation of tylosin biosynthesis in S. fradiae by ammonium ion is discussed in relation to amino acid metabolism.