Feeding experiments with [U-¹³C₃]- and (2R)-[1-²H₂]glycerol showed that glycerol is incorporated intact into carbon atoms 22, 23 and 24 of the aglycone of chlorothricin. C-1 of glycerol gives rise to C-22 with retention of one atom of deuterium, which occupies the H-22R position. A mechanism for the assembly of the aglycone is proposed which invokes phosphoenolpyruvate as the direct precursor of the 3-carbon moiety and a Baeyer-Villiger oxidation as the mode of formation of the macrocyclic lactone functionality. A feeding experiment with [1, 2-¹³C₂]succinate suggests that the propionate units of the aglycone polyketide are formed entirely via the methylmalonyl-CoA mutase reaction. The formation of the two 2, 6-dideoxy-D-rhamnose moieties of chlorothricin from glucose was shown to involve replacement of the 2-hydroxyl group of the sugar by hydrogen with inversion of configuration at C-2. This contrasts with the retention stereochemistry observed earlier for the analogous formation of the 2, 6-dideoxyhexose moiety of the antibiotic granaticin.