Biosynthesis of griseolic acids, competitive inhibitors of cyclic nucleotide phosphodiesterase, was investigated with the culture of a producing strain of Streptomyces griseoaurantiacus. C-13-Labeled and N-15-labeled compounds were added into the culture, and C-13-enriched and N-15-enriched griseolic acid A was isolated from the culture medium and analyzed by C-13 NMR and N-15 NMR spectroscopy. The compounds added to growth medium were [2-C-13]acetate, [1,2-C-13]acetate, [1,4-C-13]succinate, [1-C-13]glucose, [6-C-13]glucose, [2-C-13]ribose, and [1-C-13, N-15]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.