Biosynthesis of griseolic acids, competitive inhibitors of cyclic nucleotide phosphodiesterase, was investigated with the culture of a producing strain of Streptomyces griseoaurantiacus. 13C-Labeled and 15N-labeled compounds were added into the culture, and 13C-enriched and 15N-enriched griseolic acid A was isolated from the culture medium and analyzed by 13C NMR and 15N NMR spectroscopy. The compounds added to growth medium were [2-13C]acetate, [1,2-13C]acetate, [1,4-13C]succinate, [1-13C]glucose, [6-13C]glucose, [2-13C]ribose, and [1-13C, 15N]glycine. The results suggest that adenosine, which is formed from amino acids and sugars contributes the adenine and ribose moieties to griseolic acid A. The data also suggest that a dicarboxylic acid from the Krebs tricarboxylic acid cycle contributes to the dicarboxylic part of the compound.