All of the oxygen atoms in tylactone, a biosynthetic precursor of tylosin, are derived intact with their attached carbon atoms from C(1) of acetate, propionate, and butyrate, during its biosynthesis in cultures of a blocked mutant of Streptomyces fradiae. Tylosin is a sixteen membered ring macrolide antibiotic whose branched and highly functionalized carbon backbone is constructed from building blocks derived from acetate, propionate, butyrate, and succinate.1 The lack of detailed knowledge about the biosynthesis of such macrolide rings has recently been highlighted,2 as has the value of experiments with 13C and 18O labelled precursors which define the origins of the oxygen atoms in these and related antibiotics.3 We report below the results of our experiments using [1-13C,18O2] labelled acetate, propionate, and butyrate which demonstrate that the oxygen atoms in tylactone (1), a biosynthetic precursor of tylosin, are derived intact with their attached carbon atoms from these precursors. This provides mechanistic information about the formation of the macrolide ring and further extends the general conclusions about such metabolic pathways already accrued through biosynthetic studies on the propionate derived fourteen membered ring macrolide antibiotic erythromycin,4 the avermectins,4 and the polyether ionophore antibiotics lasalocid5 and monensin.6