The biosynthetic origin of carbonyl oxygen of uroporphyrinogen III was investigated. A model experiment using methyl benzoate labeled with 13C and 18O revealed isotope shifts of 0.015 and 0.036 ppm, corresponding to alkoxy-18O- and carbonyl-18O-labeled carbonyl carbons, in the 13C-NMR spectrum. Doubly 18O-labeled methyl benzoate showed an isotope shift of 0.050 ppm, equal to the sum of the two singly 18O-labeled carbonyl carbons. Next, [1-13C, 1,1,4-18O3]5-aminolevulinic acid (ALA) was fed to Arthrobacter hyalinus, and the resulting porphyrin was esterified with trimethyloxonium tetrafluoroborate to afford 13C- and 18O-labeled uroporphyrin III octamethyl ester. The 13C-NMR signals exhibited carbonyl carbon 18O-isotope shifts of 0.013, 0.036 and 0.051 ppm, close to those obtained in the model experiment. Thus, the carbonyl oxygens of uroporphyrinogen III and its oxidized product uroporphyrin III originated from ALA. This result shows that the loss of 18O at the acetate carbonyls of the A-ring in vitamin B12 does not occur before the formation of uroporphyrinogen III.