The biosynthesis of clavulanic acid was investigated by feeding ¹³C-labelled precursors to Streptomyces clavuligerus fermentations. The resulting samples of clavulanic acid were isolated as the benzyl ester and were examined by ¹³C NMR spectroscopy for ¹³C-enrichment. The results showed that the carbon skeleton of 1, 3-¹³C₂-glycerol was incorporated intact into the three β-lactam carbons of clavulanic acid. Studies with 1-¹³C-acetate, 2-¹³C-acetate and 1, 2-¹³C₂-acetate indicated that the remaining five carbons of clavulanic acid were probably derived from α-ketoglutarate. 1-¹³C-Propionate and 3-¹³C-propionate were not metabolised via the same route as glycerol, but were probably converted to succinate, via methylmalonyl CoA, and hence via the tricarboxylic acid cycle to the clavulanic acid precursors.